Deoxyuridine triphosphatase inhibitors

ABSTRACT

Provided herein are dUTPase inhibitors, compositions comprising such compounds and methods of using such compounds and compositions.

REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. section 119(e) to U.S.Provisional Application Ser. Nos. 61/749,791 filed Jan. 7, 2013, and61/874,643 filed Sep. 6, 2013, the content of each of which isincorporated herein in its entirety by reference.

BACKGROUND

Thymidylate metabolism is required for producing essential buildingblocks necessary to replicate DNA in dividing cells and has long been animportant therapeutic target for cornerstone cancer drugs. Drugstargeting this pathway such as 5-fluorouracil (5-FU) inhibit the enzymethymidylate synthase (TS) and are currently critical standard-of caretherapies. TS-targeted agents are heavily used for the treatment of avariety of cancers including colon, gastric, head and neck, breast, lungand blood related malignancies among others. Grem, J. L., 5-Fluorouracilplus leucovorin in cancer therapy, in Principals and Practice ofOncology Update Series, J. De Vita, V. T., S. Hellman, and A. Rosenberg,Editors. 1988, J. B. Lippincott: Philadelphia, Pa.

There are two classes of drugs that target the TS enzyme: thefluoropyrimidines and the antifolates. The fluoropyrimidines, 5 FU, S-1and capecitabine (Xeloda®), have wide use in the treatment ofgastrointestinal and breast cancers, while the antifolate pemetrexed(Alimt®) is currently used for the treatment of non-small cell lungcancer (NSCLC). Since the discovery of 5-FU over fifty years ago byCharles Heidelberger, the fluoropyrimidines remain one of the mostcommon and effective anticancer cancer drugs used worldwide. Due to thisfact, there is an abundance of clinical experience and insight into themechanism of action of these agents.

The TS inhibitor 5-fluorouracil (5 FU) remains the foundation of manyfirst and second line regimens in the treatment of colon cancer. Singleagent therapies including oxaliplatin, irinotecan, Erbitux and Avastin,demonstrate lowered activity in colon cancer as compared to 5-FU. Inaddition to colon cancer, TS-directed agents have demonstrated efficacyin several other solid tumor types.

Deoxyuridine triphosphatase (“dUTPase”) is a ubiquitous enzyme that isessential for viability in both prokaryotic and eukaryotic organisms; asthe main regulator of dUTP pools, the expression of dUTPase could haveprofound effects on the utility of chemotherapeutics that inhibitthymidylate biosynthesis. Normally, dUTPase mediates a protective roleby limiting the expansion of dUTP pools and countering the cytotoxiceffect of uracil misincorporation. According to this model, elevatedlevels of dUTPase could prevent TS inhibitor-induced dUTP accumulationand induce drug resistance. It has been shown that dUTPase overexpression results in a significant decrease in dUTP accumulation andincreased resistance to drug treatment when compared to controls.

Chemotherapeutic agents that target de novo thymidylate metabolism arecritical for the treatment of a variety of solid tumors, howeverclinical efficacy is often hindered by drug resistance. Becauseresistance to these agents is a common occurrence, the identificationand exploitation of novel determinants of drug sensitivity within thispathway of proven therapeutic utility is important. As disclosed byLadner et al. in U.S. Patent Publ. No. US 2011/0212467 (“Ladner”),uracil-DNA misincorporation pathway can play a driving role in mediatingcytotoxicity to TS-directed chemotherapies.

For example, nearly half of cancer patients do not benefit from5-FU-based treatment due to intrinsic or acquired drug resistance. Dueto this fact, there is a critical need to overcome the fundamentalchallenge of drug resistance and provide new therapeutic strategies toimprove patient outcome. This disclosure satisfies this need andprovides related advantages as well.

SUMMARY

In some aspects, this disclosure provides compounds, compositions andmethods that inhibit dUTPase when used alone or in combination with atleast one dUTPase-directed chemotherapy. In some aspects, thisdisclosure provides compounds, compositions and methods for treatingcancer, killing cancer cells, and/or inhibiting cancer cell growth whenused in combination with at least one TS-directed chemotherapy.Compounds of this class include the following compounds of formulas (I),(II), and (III).

Thus, in one aspect, provided herein are compounds of formulas (I),(II), and (III):

or a tautomer thereof, or a pharmaceutically acceptable salt of eachthereof, wherein

is a uracil isostere or a halo uracil;

is uracil, halo uracil, or a uracil isostere;W is a bond or optionally substituted —CH₂—;W¹ is a bond, N, or an optionally substituted CH group;X is a bond, O, S, NR¹⁹, optionally substituted C₁-C₆ alkylene,optionally substituted C₂-C₆ alkenylene, or optionally substituted C₂-C₆alkynylene group, a divalent optionally substituted C₆-C₁₀ aromatichydrocarbon group, or a divalent optionally substituted saturated orunsaturated C₂-C₁₀ heterocyclic or optionally substituted C₁-C₁₀heteroaryl group;R¹⁹ is hydrogen, optionally substituted C₁-C₆ alkyl or optionallysubstituted C₃-C₈ cycloalkyl;Y is a bond or an optionally substituted C₁-C₁₀ alkylene which furtheroptionally has a cycloalkylidene structure on one carbon atom, or isoptionally substituted C₂-C₆ alkenylene, or optionally substituted C₂-C₆alkynylene group, or Y is -L¹⁰-B¹-L¹¹-;L¹⁰ and L¹¹ independently are optionally substituted C₁-C₆ alkylene,optionally substituted C₂-C₆ alkenylene, or optionally substituted C₂-C₆alkynylene group;B¹ is a divalent optionally substituted C₆-C₁₀ aromatic hydrocarbongroup, or a divalent optionally substituted saturated or unsaturatedC₂-C₁₀ heterocyclic or optionally substituted C₁-C₁₀ heteroaryl group;Z is —PO₂—NR³¹R³², —SO₂NR³¹R³², —NR³PO₂—R⁴, —NR³SO₂—R⁴, or R⁴ whereinR³¹ and R³² are the same or different and each represents a hydrogenatom, optionally substituted C₁-C₆ alkyl group optionally substitutedwith an aryl group, wherein the aryl group, together with the R₁ or R₂,may form a condensed bicyclic hydrocarbon, or R³¹ and R³² are takentogether with the adjacent nitrogen atom form an optionally substitutedC₂-C₁₀ heterocyclic group or an optionally substituted C₁-C₁₀ heteroarylgroup;Z¹ is —PO₂—NR³¹R³² or —(OR³)P(O)—R⁴ wherein R³¹ and R³² areindependently a hydrogen atom, optionally substituted C₁-C₆ alkyl groupoptionally substituted with an aryl group, wherein the aryl group,together with the R³¹ or R³², may form a condensed bicyclic hydrocarbon,or R³¹ and R³² taken together with the adjacent nitrogen atom form anoptionally substituted C₂-C₁₀ heterocyclic group or an optionallysubstituted C₁-C₁₀ heteroaryl group;R³ is hydrogen or optionally substituted C₁-C₆ alkyl; andR⁴ is optionally substituted C₆-C₁₀ aryl, an optionally substitutedC₂-C₁₀ heterocyclic group, or an optionally substituted C₆-C₁₀heteroaryl group.

This disclosure also provides a tautotomer, or its pharmaceuticallyacceptable salt of a compound as disclosed herein. Methods to preparesuch are known in the art.

This disclosure also provides a stereochemically pure enantiomer of acompound as described herein, its tautotomer, diastereoisomer or itspharmaceutically acceptable salt. Methods to purify and identify thepure enantiomer are known in the art and described herein.

In one aspect, the compound is provided as a stereochemically pureenantiomer, e.g., PCI 10586, as described herein. Pharmaceuticallyacceptable salts of PCI 10586 are also provided herein.

In another aspect, compositions comprising one or more of theabove-noted compounds and a carrier are provided. In one embodiment, thecomposition is a pharmaceutical composition and therefore furthercomprises at least a pharmaceutically acceptable carrier or apharmaceutically acceptable excipient. The compositions are formulatedfor various delivery modes, e.g., systemic (oral) or local.

In another aspect, this disclosure provides compositions comprising oneor more compounds as provided herein and a dUTPase-directed chemotherapyand a carrier, such as a pharmaceutically acceptable carrier. Thecompound and chemotherapy can be in varying amounts, and in one aspect,each in an effective amount when used in combination, provides atherapeutic benefit as described herein. The compositions are formulatedfor various delivery modes, e.g., systemic (oral) or local.

In another aspect, methods are provided for inhibiting deoxyuridinetriphosphatase (dUTPase) comprising contacting the dUTPase with aneffective amount of a compound or a composition provided herein. Inanother aspect, the method further comprises contacting the dUTPase witha dUTPase-directed chemotherapy alone or in combination with thecompound as provided herein. The contacting can be simultaneous orconcurrent. In a further aspect the dUTPase-directed chemotherapy iscontacted prior to the compound or composition as described herein. Inanother aspect, the dUTPase-directed chemotherapy is contactedsubsequent to the compound or composition. In a yet further aspect, thecompound or composition and the dUTPase-directed chemotherapy aresequentially administered through several rounds of therapy. Thecontacting can be simultaneous or concurrent and/or in vitro (cellfree), ex vivo or in vivo. In a further aspect, the compounds orcompositions of this disclosure are administered to a patient identifiedor selected for the therapy by determining that the patient has a tumoror mass that over expresses dUTPase. Methods to identify such patientsare known in the art and incorporated herein. The methods whenadministered to a subject such as a human patient, can be first line,second line, third line, forth line or further therapy.

Also provided is a method for reversing resistance to a dUTPase-directedchemotherapy comprising contacting a cell over expressing dUTPase withan effective amount of a compound or a composition provided herein,alone or in combination with a dUTPase-directed chemotherapy. In oneaspect, the cell is first identified as over expressing dUTPase by ascreen as disclosed by U.S. Pat. No. 5,962,246. In another aspect, themethod further comprises subsequently contacting the cell expressingdUTPase with a dUTPase-directed chemotherapy. The methods can beadministered as second line, third line, forth line or further therapy.

Further provided is a method for enhancing the efficacy of adUTPase-directed chemotherapy comprising contacting a cell, e.g., in oneaspect a over expressing dUTPase, with an effective amount of a compoundor a composition provided herein. In another aspect, the method furthercomprises contacting the cell with a dUTPase-directed chemotherapy. Thecontacting can be simultaneous or concurrent and/or in vitro (cellfree), ex vivo or in vivo. In a further aspect the dUTPase-directedchemotherapy is contacted prior to the compound or composition asdescribed herein, or vice versa. The methods when administered to asubject such as a human patient, can be first line, second line, thirdline, forth line or further therapy.

In another aspect, provided herein is a method of treating a diseaseassociated with the dUTPase pathway, e.g., cancer, viral infection,bacterial infection, or an autoimmune disorder, comprising administeringto a patient in need of such treatment an effective amount of thecompound provided herein or a composition provided herein in combinationwith an agent which is suitable for treating the disease, therebytreating the disease. The administration of the compound of thisinvention and the agent that is suitable for the disease (e.g., adUTPase inhibitor) can be simultaneous or concurrent and/or in vitro(cell free), ex vivo or in vivo. In a further aspect the agent that issuitable for treating the disease is administered prior to the compoundor composition as described herein, or vice versa. In one aspect, thepatient being treated is selected for the therapy by screening a cell ortissue sample isolated from the patient for over expression of dUTPase.The therapy is then administered to this patient after the screen.

In another aspect, provided herein is a kit comprising a compoundprovided herein or a composition provided herein and one more of adUTPase inhibitor (e.g., an antitumor agent) and instructions foradministering the agent. Yet further provided in the kit are reagentsand instructions to screen for dUTPase expression.

In each of the above embodiments, a non-limiting example of the dUTPasemediated chemotherapy comprises a TS-inhibitor, e.g., 5-FU or 5-FUcontaining therapy such as 5-FU based adjuvant therapy and chemicalequivalents thereof.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A and B show characterization of PCI 10213 by (A) HPLC (A1) andMS (A2) and (B) ¹H-NMR.

FIGS. 2A and B show characterization of PCI 10214 by (A) HPLC (A1) andMS (A2) (B) ¹H-NMR.

FIGS. 3A-C show in (A) dUTPase enzyme inhibition assay showing %inhibition at increasing concentrations of PCI 10213 and PCI 10216, andin (B) and (C), MTS assays where colon cancer and NSCLC cancer cellswere treated with PCI 10213, PCI 10214 and PCI 10216 alone. Data ispresented as % control of vehicle-treated controls.

FIGS. 4A and B show MTS assay where HCT116 (A) and SW620 (B) coloncancer cells were treated with a fixed dose of 25 μmol/L PCI 10213 orPCI 10216 alone and in combination with increasing doses of 5-FU. Datais presented as % control of vehicle-treated controls.

FIG. 5 shows a colony formation assay, where NSCLC, colon and breastcancer cells were treated with PCI 10213 alone and in combination with afixed dose of FUdR. Data is presented as % control of vehicle-treatedcontrols. Bars represent mean±SEM. Representative images for one NSCLC,one colon and one breast cancer cell line are showing in FIGS. 6 and 7.

FIGS. 6A-6D show a colony formation assay, where HCT116 colon cancercells were treated with PCI 10213, PCI 10214, PCI 10216 alone and incombination with a fixed dose of FUdR. Representative images are scansof the colonies stained with crystal violet. (A) Cells treated withincreasing concentrations of FUdR alone. (B) Cells treated withincreasing concentrations of PCI 10213 alone (top row) and combinationwith 0.5 μmol/L FUdR. (C) Cells treated with increasing concentrationsof PCI 10214 alone (top row) and combination with 0.5 μmol/L FUdR. (D)Cells treated with increasing concentrations of PCI 10216 alone (toprow) and combination with 0.5 μmol/L FUdR.

FIGS. 7A-C show colony formation assay, where A549 NSCLC cells weretreated with PCI 10213 or PCI 10216 alone and in combination with afixed dose of FUdR. Representative images are scans of the coloniesstained with crystal violet. (A) Cells treated with increasingconcentrations of FUdR alone. (B) Cells treated with increasingconcentrations of PCI 10213 alone (top row) and combination with 0.5μmol/L FUdR. (C) Cells treated with increasing concentrations of PCI10216 alone (top row) and combination with 0.5 μmol/L FUdR.

FIGS. 8A-C show a colony formation assay, where MCF7 breast cancer cellswere treated with PCI 10213 or PCI 10216 alone and in combination with afixed dose of FUdR. Representative images are scans of the coloniesstained with crystal violet. (A) Cells treated with increasingconcentrations of FUdR alone. (B) Cells treated with increasingconcentrations of PCI 10213 alone (top row) and combination with 0.5μmol/L FUdR. (C) Cells treated with increasing concentrations of PCI10216 alone (top row) and combination with 0.5 μmol/L FUdR.

FIG. 9 shows HPLC chromatogram of PCI 10586 with retention time(R_(t))=28.4 min.

FIG. 10 shows HPLC chromatogram of PCI 10585 with R_(t)=22.13 min.

FIGS. 11A-11C show the results of a colony forming assay. HCT116 coloncancer cells were treated with PCI 10213, PCI 10585, PCI 102586 aloneand in combination with a fixed dose of FUdR. Representative images arescans of the colonies stained with crystal violet. (A) Cells treatedwith increasing concentrations of FUdR alone. (B) Cells treated withincreasing concentrations of PCI 10213, 10585, 10586 alone. (C) Cellstreated with increasing concentrations of PCI 10213, 10585 and 10586 incombination with 0.5 μmol/L FUdR.

FIG. 12 graphically shows quantitation of a colony formation assay.Briefly, HCT116 colon cancer cells were treated with PCI 10213, PCI10585, PCI 102586 alone and in combination with a fixed dose of 0.5μmol/L FUdR. Bars represent the number of colonies counted followingstaining with crystal violet. Top, PCI 10213; middle, PCI 10585; bottom,PCI 10586.

DETAILED DESCRIPTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure in their entirety to more fully describe the state of the artto which this invention pertains.

DEFINITIONS

The practice of the present technology will employ, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,immunology, molecular biology, microbiology, cell biology andrecombinant DNA, which are within the skill of the art. See, e.g.,Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual,2^(nd) edition (1989); Current Protocols In Molecular Biology (F. M.Ausubel, et al. eds., (1987)); the series Methods in Enzymology(Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson,B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988)Antibodies, a Laboratory Manual, and Animal Cell Culture (R. I.Freshney, ed. (1987)).

As used in the specification and claims, the singular form “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludeothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants, e.g., from the isolation and purification method andpharmaceutically acceptable carriers, preservatives, and the like.“Consisting of” shall mean excluding more than trace elements of otheringredients. Embodiments defined by each of these transition terms arewithin the scope of this technology.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1, 5, or 10%. It is to be understood,although not always explicitly stated that all numerical designationsare preceded by the term “about.” It also is to be understood, althoughnot always explicitly stated, that the reagents described herein aremerely exemplary and that equivalents of such are known in the art.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms.This term includes, by way of example, linear and branched hydrocarbylgroups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—),isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—),sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—), n-pentyl(CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

“Alkenyl” refers to monovalent straight or branched hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of vinyl (>C═C<)unsaturation. Such groups are exemplified, for example, by vinyl, allyl,and but-3-en-1-yl. Included within this term are the cis and transisomers or mixtures of these isomers.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of acetylenic (—C≡C—)unsaturation. Examples of such alkynyl groups include acetylenyl(—C═CH), and propargyl (—CH₂C≡CH).

“Substituted alkyl” refers to an alkyl group having from 1 to 5,preferably 1 to 3, or more preferably 1 to 2 substituents selected fromthe group consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy,substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substitutedcycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substitutedheteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio,substituted heteroarylthio, heterocyclic, substituted heterocyclic,heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,substituted heterocyclylthio, nitro, SO₃H, substituted sulfonyl,substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substitutedalkylthio, wherein said substituents are as defined herein.

“Substituted alkenyl” refers to alkenyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy,substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substitutedcycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,guanidino, substituted guanidino, halo, hydroxyl, heteroaryl,substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy,heteroarylthio, substituted heteroarylthio, heterocyclic, substitutedheterocyclic, heterocyclyloxy, substituted heterocyclyloxy,heterocyclylthio, substituted heterocyclylthio, nitro, SO₃H, substitutedsulfonyl, substituted sulfonyloxy, thioacyl, thiol, alkylthio, andsubstituted alkylthio, wherein said substituents are as defined hereinand with the proviso that any hydroxyl or thiol substitution is notattached to a vinyl (unsaturated) carbon atom.

“Substituted alkynyl” refers to alkynyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy,substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substitutedcycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substitutedheteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio,substituted heteroarylthio, heterocyclic, substituted heterocyclic,heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,substituted heterocyclylthio, nitro, SO₃H, substituted sulfonyl,substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substitutedalkylthio, wherein said substituents are as defined herein and with theproviso that any hydroxyl or thiol substitution is not attached to anacetylenic carbon atom.

“Alkylene” refers to divalent saturated aliphatic hydrocarbyl groupspreferably having from 1 to 6 and more preferably 1 to 3 carbon atomsthat are either straight-chained or branched. This term is exemplifiedby groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)— or —CH(CH₃)CH₂—), butylene(—CH₂CH₂CH₂CH₂—), isobutylene (—CH₂CH(CH₃)CH₂—), sec-butylene(—CH₂CH₂(CH₃)CH—), and the like. Similarly, “alkenylene” and“alkynylene” refer to an alkylene moiety containing respective 1 or 2carbon carbon double bonds or a carbon carbon triple bond.

“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents selected from the group consistingof alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl,substituted aryl, aryloxy, substituted aryloxy, cyano, halogen,hydroxyl, nitro, carboxyl, carboxyl ester, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic,substituted heterocyclic, and oxo wherein said substituents are definedherein.

In some embodiments, the alkylene has 1 to 2 of the aforementionedgroups, or having from 1-3 carbon atoms replaced with —O—, —S—, or—NR^(Q)— moieties where R^(Q) is H or C₁-C₆ alkyl. It is to be notedthat when the alkylene is substituted by an oxo group, 2 hydrogensattached to the same carbon of the alkylene group are replaced by “═O”.“Substituted alkenylene“and” substituted alkynylene” refer to alkenyleneand substituted alkynylene moieties substituted with substituents asdescribed for substituted alkylene.

“Alkoxy” refers to the group —O-alkyl wherein alkyl is defined herein.Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and n-pentoxy.

“Substituted alkoxy” refers to the group —O-(substituted alkyl) whereinsubstituted alkyl is defined herein.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O)—, heterocyclic-C(O)—, and substitutedheterocyclic-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein. Acyl includes the“acetyl” group CH₃C(O)—.

“Acylamino” refers to the groups —NR⁴⁷C(O)alkyl, —NR⁴⁷C(O) substitutedalkyl, —NR⁴⁷C(O)cycloalkyl, —NR⁴⁷C(O) substituted cycloalkyl,—NR⁴⁷C(O)cycloalkenyl, —NR⁴⁷C(O) substituted cycloalkenyl,—NR⁴⁷C(O)alkenyl, —NR⁴⁷C(O) substituted alkenyl, —NR⁴⁷C(O)alkynyl,—NR⁴⁷C(O) substituted alkynyl, —NR⁴⁷C(O)aryl, —NR⁴⁷C(O) substitutedaryl, —NR⁴⁷C(O)heteroaryl, —NR⁴⁷C(O) substituted heteroaryl,—NR⁴⁷C(O)heterocyclic, and —NR⁴⁷C(O) substituted heterocyclic whereinR⁴⁷ is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—,alkenyl-C(O)O—, substituted alkenyl-C(O)O—, alkynyl-C(O)O—, substitutedalkynyl-C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—,substituted cycloalkyl-C(O)O—, cycloalkenyl-C(O)O—, substitutedcycloalkenyl-C(O)O—, heteroaryl-C(O)O—, substituted heteroaryl-C(O)O—,heterocyclic-C(O)O—, and substituted heterocyclic-C(O)O— wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

An animal, subject or patient for diagnosis or treatment refers to ananimal such as a mammal, or a human, ovine, bovine, feline, canine,equine, simian, etc. Non-human animals subject to diagnosis or treatmentinclude, for example, simians, murine, such as, rat, mice, canine,leporid, livestock, sport animals, and pets.

“Amino” refers to the group —NH₂.

“Substituted amino” refers to the group —NR⁴⁸R⁴⁹ where R⁴⁸ and R⁴⁹ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl,—SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-cycloalkenyl,—SO₂-substituted cylcoalkenyl, —SO₂-aryl, —SO₂-substituted aryl,—SO₂-heteroaryl, —SO₂-substituted heteroaryl, —SO₂-heterocyclic, and—SO₂-substituted heterocyclic and wherein R⁴⁸ and R⁴⁹ are optionallyjoined, together with the nitrogen bound thereto to form a heterocyclicor substituted heterocyclic group, provided that R⁴⁸ and R⁴⁹ are bothnot hydrogen, and wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein. When R⁴⁸ is hydrogen and R⁴⁹ isalkyl, the substituted amino group is sometimes referred to herein asalkylamino. When R⁴⁸ and R⁴⁹ are alkyl, the substituted amino group issometimes referred to herein as dialkylamino. When referring to amonosubstituted amino, it is meant that either R⁴⁸ or R⁴⁹ is hydrogenbut not both. When referring to a disubstituted amino, it is meant thatneither R⁴⁸ nor R⁴⁹ are hydrogen.

“Aminocarbonyl” refers to the group —C(O)NR⁵⁰R^(5′) where R⁵⁰ and R⁵¹are independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R⁵⁰ andR⁵¹ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Aminothiocarbonyl” refers to the group —C(S)NR⁵⁰R⁵¹ where R⁵⁰ and R⁵¹are independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R⁵⁰ andR⁵¹ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Aminocarbonylamino” refers to the group —NR⁴⁷C(O)NR⁵⁰R⁵¹ where R⁴⁷ ishydrogen or alkyl and R⁵⁰ and R⁵¹ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic, and where R⁵⁰ and R⁵¹ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminothiocarbonylamino” refers to the group —NR⁴⁷C(S)NR⁵⁰R⁵¹ where R⁴⁷is hydrogen or alkyl and R⁵⁰ and R⁵¹ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R⁵⁰ and R⁵¹ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonyloxy” refers to the group —O—C(O)NR⁵⁰R⁵¹ where R⁵⁰ and R⁵¹are independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R⁵⁰ andR⁵¹ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Aminosulfonyl” refers to the group —SO₂NR⁵⁰R⁵¹ where R⁵⁰ and R⁵¹ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R⁵⁰ andR⁵¹ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Aminosulfonyloxy” refers to the group —O—SO₂NR⁵⁰R⁵¹ where R⁵⁰ and R⁵¹are independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R⁵⁰ andR⁵¹ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Aminosulfonylamino” refers to the group —NR⁴⁷SO₂NR⁵⁰R⁵¹ where R⁴⁷ ishydrogen or alkyl and R⁵⁰ and R⁵¹ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R⁵⁰ and R⁵¹ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Amidino” refers to the group —C(═NR⁵²)NR⁵⁰R⁵¹ where R⁵⁰, R⁵¹, and R⁵²are independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R⁵⁰ andR⁵¹ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is at an aromatic carbon atom. Preferred aryl groupsinclude phenyl and naphthyl.

“Substituted aryl” refers to aryl groups which are substituted with 1 to5, preferably 1 to 3, or more preferably 1 to 2 substituents selectedfrom the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl,aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy,cycloalkenylthio, substituted cycloalkenylthio, guanidino, substitutedguanidino, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substitutedheteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy,substituted heterocyclyloxy, heterocyclylthio, substitutedheterocyclylthio, nitro, SO₃H, substituted sulfonyl, substitutedsulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,wherein said substituents are as defined herein.

“Aryloxy” refers to the group —O-aryl, where aryl is as defined herein,that includes, by way of example, phenoxy and naphthoxy.

“Substituted aryloxy” refers to the group —O-(substituted aryl) wheresubstituted aryl is as defined herein.

“Arylthio” refers to the group —S-aryl, where aryl is as defined herein.

“Substituted arylthio” refers to the group —S-(substituted aryl), wheresubstituted aryl is as defined herein.

“Carbonyl” refers to the divalent group —C(O)— which is equivalent to—C(═O)—.

“Carboxyl” or “carboxy” refers to —COOH or salts thereof.

“Carboxyl ester” or “carboxy ester” refers to the groups —C(O)O-alkyl,—C(O)O-substituted alkyl, —C(O)O-alkenyl, —C(O)O-substituted alkenyl,—C(O)O-alkynyl, —C(O)O-substituted alkynyl, —C(O)O-aryl,—C(O)O-substituted aryl, —C(O)O-cycloalkyl, —C(O)O-substitutedcycloalkyl, —C(O)O-cycloalkenyl, —C(O)O-substituted cycloalkenyl,—C(O)O-heteroaryl, —C(O)O-substituted heteroaryl, —C(O)O-heterocyclic,and —C(O)O-substituted heterocyclic wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“(Carboxyl ester)amino” refers to the group —NR⁴⁷C(O)O-alkyl,—NR⁴⁷C(O)O-substituted alkyl, —NR⁴⁷C(O)O-alkenyl, —NR⁴⁷C(O)O-substitutedalkenyl, —NR⁴⁷C(O)O-alkynyl, —NR⁴⁷C(O)O-substituted alkynyl,—NR⁴⁷C(O)O-aryl, —NR⁴⁷C(O)O-substituted aryl, —NR⁴⁷C(O)O-cycloalkyl,—NR⁴⁷C(O)O-substituted cycloalkyl, —NR⁴⁷C(O)O-cycloalkenyl,—NR⁴⁷C(O)O-substituted cycloalkenyl, —NR⁴⁷C(O)O-heteroaryl,—NR⁴⁷C(O)O-substituted heteroaryl, —NR⁴⁷C(O)O-heterocyclic, and—NR⁴⁷C(O)O-substituted heterocyclic wherein R⁴⁷ is alkyl or hydrogen,and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein.

“(Carboxyl ester)oxy” refers to the group —O—C(O)O-alkyl,—O—C(O)O-substituted alkyl, —O—C(O)O-alkenyl, —O—C(O)O-substitutedalkenyl, —O—C(O)O-alkynyl, —O—C(O)O-substituted alkynyl, —O—C(O)O-aryl,—O—C(O)O-substituted aryl, —O—C(O)O-cycloalkyl, —O—C(O)O-substitutedcycloalkyl, —O—C(O)O-cycloalkenyl, —O—C(O)O-substituted cycloalkenyl,—O—C(O)O-heteroaryl, —O—C(O)O-substituted heteroaryl,—O—C(O)O-heterocyclic, and —O—C(O)O-substituted heterocyclic whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

A “composition” as used herein, intends an active agent, such as acompound as disclosed herein and a carrier, inert or active. The carriercan be, without limitation, solid such as a bead or resin, or liquid,such as phosphate buffered saline.

Administration or treatment in “combination” refers to administering twoagents such that their pharmacological effects are manifest at the sametime. Combination does not require administration at the same time orsubstantially the same time, although combination can include suchadministrations.

“Cyano” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspiro ring systems. The fused ring can be an aryl ring provided that thenon aryl part is joined to the rest of the molecule. Examples ofsuitable cycloalkyl groups include, for instance, adamantyl,cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl.

“Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to10 carbon atoms having single or multiple cyclic rings and having atleast one >C═C< ring unsaturation and preferably from 1 to 2 sitesof >C═C< ring unsaturation.

“Substituted cycloalkyl” and “substituted cycloalkenyl” refers to acycloalkyl or cycloalkenyl group having from 1 to 5 or preferably 1 to 3substituents selected from the group consisting of oxo, thioxo, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl,aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy,cycloalkenylthio, substituted cycloalkenylthio, guanidino, substitutedguanidino, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substitutedheteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy,substituted heterocyclyloxy, heterocyclylthio, substitutedheterocyclylthio, nitro, SO₃H, substituted sulfonyl, substitutedsulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,wherein said substituents are as defined herein.

“Cycloalkyloxy” refers to —O-cycloalkyl.

“Substituted cycloalkyloxy refers to —O-(substituted cycloalkyl).

“Cycloalkylthio” refers to —S-cycloalkyl.

“Substituted cycloalkylthio” refers to —S-(substituted cycloalkyl).

“Cycloalkenyloxy” refers to —O-cycloalkenyl.

“Substituted cycloalkenyloxy” refers to —O-(substituted cycloalkenyl).

“Cycloalkenylthio” refers to —S-cycloalkenyl.

“Substituted cycloalkenylthio” refers to —S-(substituted cycloalkenyl).

“Guanidino” refers to the group —NHC(═NH)NH₂.

“Substituted guanidino” refers to —NR⁵³C(═NR⁵³)N(R⁵³)₂ where each R⁵³ isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, andsubstituted heterocyclic and two R⁵³ groups attached to a commonguanidino nitrogen atom are optionally joined together with the nitrogenbound thereto to form a heterocyclic or substituted heterocyclic group,provided that at least one R⁵³ is not hydrogen, and wherein saidsubstituents are as defined herein.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur within the ring. Such heteroaryl groups can have asingle ring (e.g., pyridinyl or furyl) or multiple condensed rings(e.g., indolizinyl or benzothienyl) wherein the condensed rings may ormay not be aromatic and/or contain a heteroatom provided that the pointof attachment is through an atom of the aromatic heteroaryl group. Inone embodiment, the nitrogen and/or the sulfur ring atom(s) of theheteroaryl group are optionally oxidized to provide for the N-oxide(N→O), sulfinyl, or sulfonyl moieties. Certain non-limiting examplesinclude pyridinyl, pyrrolyl, indolyl, thiophenyl, oxazolyl, thizolyl,and furanyl.

“Substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to2 substituents selected from the group consisting of the same group ofsubstituents defined for substituted aryl.

“Heteroaryloxy” refers to —O-heteroaryl.

“Substituted heteroaryloxy” refers to the group —O-(substitutedheteroaryl).

“Heteroarylthio” refers to the group —S-heteroaryl.

“Substituted heteroarylthio” refers to the group —S-(substitutedheteroaryl).

“Heterocycle” or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl”refers to a saturated or partially saturated, but not aromatic, grouphaving from 1 to 10 ring carbon atoms and from 1 to 4 ring heteroatomsselected from the group consisting of nitrogen, sulfur, or oxygen.Heterocycle encompasses single ring or multiple condensed rings,including fused bridged and spiro ring systems. In fused ring systems,one or more the rings can be cycloalkyl, aryl, or heteroaryl providedthat the point of attachment is through a non-aromatic ring. In oneembodiment, the nitrogen and/or sulfur atom(s) of the heterocyclic groupare optionally oxidized to provide for the N-oxide, sulfinyl, orsulfonyl moieties.

“Substituted heterocyclic” or “substituted heterocycloalkyl” or“substituted heterocyclyl” refers to heterocyclyl groups that aresubstituted with from 1 to 5 or preferably 1 to 3 of the samesubstituents as defined for substituted cycloalkyl.

“Heterocyclyloxy” refers to the group —O-heterocycyl.

“Substituted heterocyclyloxy” refers to the group —O-(substitutedheterocycyl).

“Heterocyclylthio” refers to the group —S-heterocycyl.

“Substituted heterocyclylthio” refers to the group —S-(substitutedheterocycyl).

Examples of heterocycle and heteroaryls include, but are not limited to,azetidine, pyrrole, furan, thiophene, imidazole, pyrazole, pyridine,pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine,phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine,phenothiazine, imidazolidine, imidazoline, piperidine, piperazine,indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,and tetrahydrofuranyl.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O).

Phenylene refers to a divalent aryl ring, where the ring contains 6carbon atoms.

Substituted phenylene refers to phenylenes which are substituted with 1to 4, preferably 1 to 3, or more preferably 1 to 2 substituents selectedfrom the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl,aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy,cycloalkenylthio, substituted cycloalkenylthio, guanidino, substitutedguanidino, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substitutedheteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy,substituted heterocyclyloxy, heterocyclylthio, substitutedheterocyclylthio, nitro, SO₃H, substituted sulfonyl, substitutedsulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,wherein said substituents are as defined herein.

“Spirocycloalkyl” and “spiro ring systems” refers to divalent cyclicgroups from 3 to 10 carbon atoms having a cycloalkyl or heterocycloalkylring with a spiro union (the union formed by a single atom which is theonly common member of the rings) as exemplified by the followingstructure:

“Sulfonyl” refers to the divalent group —S(O)₂—.

“Substituted sulfonyl” refers to the group —SO₂-alkyl, —SO₂-substitutedalkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl, —SO₂-cycloalkyl,—SO₂-substituted cycloalkyl, —SO₂-cycloalkenyl, —SO₂-substitutedcylcoalkenyl, —SO₂-aryl, —SO₂-substituted aryl, —SO₂-heteroaryl,—SO₂-substituted heteroaryl, —SO₂-heterocyclic, —SO₂-substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein. Substituted sulfonyl includes groupssuch as methyl-SO₂—, phenyl-SO₂—, and 4-methylphenyl-SO₂—.

“Substituted sulfonyloxy” refers to the group —OSO₂-alkyl,—OSO₂-substituted alkyl, —OSO₂-alkenyl, —OSO₂-substituted alkenyl,—OSO₂-cycloalkyl, —OSO₂-substituted cycloalkyl, —OSO₂-cycloalkenyl,—OSO₂-substituted cylcoalkenyl, —OSO₂-aryl, —OSO₂-substituted aryl,—OSO₂-heteroaryl, —OSO₂-substituted heteroaryl, —OSO₂-heterocyclic,—OSO₂-substituted heterocyclic, wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Thioacyl” refers to the groups H—C(S)—, alkyl-C(S)—, substitutedalkyl-C(S)—, alkenyl-C(S)—, substituted alkenyl-C(S)—, alkynyl-C(S)—,substituted alkynyl-C(S)—, cycloalkyl-C(S)—, substitutedcycloalkyl-C(S)—, cycloalkenyl-C(S)—, substituted cycloalkenyl-C(S)—,aryl-C(S)—, substituted aryl-C(S)—, heteroaryl-C(S)—, substitutedheteroaryl-C(S)—, heterocyclic-C(S)—, and substitutedheterocyclic-C(S)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Thiol” refers to the group —SH.

“Thiocarbonyl” refers to the divalent group —C(S)— which is equivalentto —C(═S)—.

“Thioxo” refers to the atom (═S).

“Alkylthio” refers to the group —S-alkyl wherein alkyl is as definedherein.

“Substituted alkylthio” refers to the group —S-(substituted alkyl)wherein substituted alkyl is as defined herein.

“Optionally substituted” refers to a group selected from that group anda substituted form of that group. Substituted groups are defined herein.In one embodiment, substituents are selected from C₁-C₁₀ or C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₃-C₈ cycloalkyl, C₂-C₁₀heterocyclyl, C₁-C₁₀ heteroaryl, halo, nitro, cyano, —CO₂H or a C₁-C₆alkyl ester thereof.

“Tautomer” refer to alternate forms of a compound that differ in theposition of a proton, such as enol-keto and imine-enamine tautomers, orthe tautomeric forms of heteroaryl groups containing a ring atomattached to both a ring —NH— moiety and a ring ═N— moiety such aspyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles.

“Uracil isostere” refers to an isostere of uracil. Such a moietyprovides some or all of the hydrogen bond acceptor-donor-acceptorproperty of uracil and optionally provides other structuralcharacteristics of uracil. A skilled artisan will further appreciate themeaning of this term by reading the non limiting examples of such uracilisosteres provided herein.

As used herein, the term stereochemically pure denotes a compound whichhas 80% or greater by weight of the indicated stereoisomer and 20% orless by weight of other stereoisomers. In a further embodiment, thecompound of formula (I), (II), or (III) has 90% or greater by weight ofthe stated stereoisomer and 10% or less by weight of otherstereoisomers. In a yet further embodiment, the compound of formula (I)has 95% or greater by weight of the stated stereoisomer and 5% or lessby weight of other stereoisomers. In a still further embodiment, thecompound of formula (I), (II), or (III) has 97% or greater by weight ofthe stated stereoisomer and 3% or less by weight of other stereoisomers.

“Pharmaceutically acceptable salt” refers to salts of a compound, whichsalts are suitable for pharmaceutical use and are derived from a varietyof organic and inorganic counter ions well known in the art and include,when the compound contains an acidic functionality, by way of exampleonly, sodium, potassium, calcium, magnesium, ammonium, andtetraalkylammonium; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, andoxalate (see Stahl and Wermuth, eds., “Handbook of PharmaceuticallyAcceptable Salts,” (2002), Verlag Helvetica Chimica Acta, Zürich,Switzerland), for a discussion of pharmaceutical salts, their selection,preparation, and use.

Generally, pharmaceutically acceptable salts are those salts that retainsubstantially one or more of the desired pharmacological activities ofthe parent compound and which are suitable for in vivo administration.Pharmaceutically acceptable salts include acid addition salts formedwith inorganic acids or organic acids. Inorganic acids suitable forforming pharmaceutically acceptable acid addition salts include, by wayof example and not limitation, hydrohalide acids (e.g., hydrochloricacid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid, nitricacid, phosphoric acid, and the like.

Organic acids suitable for forming pharmaceutically acceptable acidaddition salts include, by way of example and not limitation, aceticacid, trifluoroacetic acid, propionic acid, hexanoic acid,cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,alkylsulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid,1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, etc.),arylsulfonic acids (e.g., benzenesulfonic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, etc.), glutamic acid, hydroxynaphthoic acid,salicylic acid, stearic acid, muconic acid, and the like.

Pharmaceutically acceptable salts also include salts formed when anacidic proton present in the parent compound is either replaced by ametal ion (e.g., an alkali metal ion, an alkaline earth metal ion, or analuminum ion) or by an ammonium ion (e.g., an ammonium ion derived froman organic base, such as, ethanolamine, diethanolamine, triethanolamine,morpholine, piperidine, dimethylamine, diethylamine, triethylamine, andammonia).

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages. Such delivery is dependent ona number of variables including the time period for which the individualdosage unit is to be used, the bioavailability of the therapeutic agent,the route of administration, etc. It is understood, however, thatspecific dose levels of the therapeutic agents disclosed herein for anyparticular subject depends upon a variety of factors including theactivity of the specific compound employed, bioavailability of thecompound, the route of administration, the age of the animal and itsbody weight, general health, sex, the diet of the animal, the time ofadministration, the rate of excretion, the drug combination, and theseverity of the particular disorder being treated and form ofadministration. In general, one will desire to administer an amount ofthe compound that is effective to achieve a serum level commensuratewith the concentrations found to be effective in vivo. Theseconsiderations, as well as effective formulations and administrationprocedures are well known in the art and are described in standardtextbooks. Consistent with this definition and as used herein, the term“therapeutically effective amount” is an amount sufficient to treat aspecified disorder or disease or alternatively to obtain apharmacological response such as inhibiting dUTPase.

As used herein, “treating” or “treatment” of a disease in a patientrefers to (1) preventing the symptoms or disease from occurring in ananimal that is predisposed or does not yet display symptoms of thedisease; (2) inhibiting the disease or arresting its development; or (3)ameliorating or causing regression of the disease or the symptoms of thedisease. As understood in the art, “treatment” is an approach forobtaining beneficial or desired results, including clinical results. Forthe purposes of this technology, beneficial or desired results caninclude one or more, but are not limited to, alleviation or ameliorationof one or more symptoms, diminishment of extent of a condition(including a disease), stabilized (i.e., not worsening) state of acondition (including disease), delay or slowing of condition (includingdisease), progression, amelioration or palliation of the condition(including disease), states and remission (whether partial or total),whether detectable or undetectable.

“dUTPase” means any of the following, which are considered to besynonymous, “deoxyuridine triphosphate nucleotidohydrolase”,“deoxyuridine triphosphate pyrophosphatase”, “dUTP nucleotidohydrolase”,“dUTP pyrophosphatase”, and other equivalent nomenclature for thedUTPase enzyme. In one aspect, dUTPase intends DUT-N and DUT-M. In otheraspects, it is DUT-N only, or alternatively, DUT-M only. The amino acidand coding sequences for dUTPase are known in the art and disclosed inU.S. Pat. No. 5,962,246. Methods for expressing and screening forexpression level of the enzyme are disclosed in U.S. Pat. No. 5,962,246and Ladner et al. (US Patent Publ. No. 2011/0212467A1).

“DUT-N” means the nuclear form of dUTPase.

“DUT-M” means the mitochondrial or cytoplasmic form of dUTPase.

“dUTPase-directed therapy” intends therapeutics that target the dUTPasepathway, e.g., in the case of cancer, e.g. TS-directed therapies and thefluoropyrimidines (such as 5-FU), pemetrexed (Alimta®), capecitabine(Xeloda®), S-1 and antifolates (such as methotrexate) and chemicalequivalents thereof. Non-limiting examples include 5-flurouracil (5-FU),TS-directed therapies and 5-FU based adjuvant therapy. Combinationtherapies can include any intervention that alters nucleotide poolsand/or sensitizes the immune cells or viruses to the dUTPase inhibitor,as are well known to the skilled artisan. For rheumatoid arthritis, forexample, the combination can be with an dihydrofolate reductase (DHFR)inhibitor such as methotrexate.

5-fluorouracil (5-FU) belongs to the family of therapy drugs calledpyrimidine based anti-metabolites. It is a pyrimidine analog, which istransformed into different cytotoxic metabolites that are thenincorporated into DNA and RNA thereby inducing cell cycle arrest andapoptosis. Chemical equivalents are pyrimidine analogs which result indisruption of DNA replication. Chemical equivalents inhibit cell cycleprogression at S phase resulting in the disruption of cell cycle andconsequently apoptosis. Equivalents to 5-FU include prodrugs, analogsand derivative thereof such as 5′-deoxy-5-fluorouridine(doxifluoroidine), 1-tetrahydrofuranyl-5-fluorouracil (ftorafur),capecitabine (Xeloda®), S-1 (MBMS-247616, consisting of tegafur and twomodulators, a 5-chloro-2,4-dihydroxypyridine and potassium oxonate),ralititrexed (tomudex), nolatrexed (Thymitaq, AG337), LY231514 andZD9331, as described for example in Papamicheal (1999) The Oncologist4:478-487.

“5-FU based adjuvant therapy” refers to 5-FU alone or alternatively thecombination of 5-FU with other treatments, that include, but are notlimited to radiation, methyl-CCNU, leucovorin, oxaliplatin, irinotecin,mitomycin, cytarabine, levamisole. Specific treatment adjuvant regimensare known in the art as FOLFOX, FOLFOX4, FOLFIR1, MOF (semustine(methyl-CCNU), vincrisine (Oncovin®) and 5-FU). For a review of thesetherapies see Beaven and Goldberg (2006) Oncology 20(5):461-470. Anexample of such is an effective amount of 5-FU and Leucovorin. Otherchemotherapeutics can be added, e.g., oxaliplatin or irinotecan.

Capecitabine is a prodrug of (5-FU) that is converted to its active formby the tumor-specific enzyme PynPase following a pathway of threeenzymatic steps and two intermediary metabolites,5′-deoxy-5-fluorocytidine (5′-DFCR) and 5′-deoxy-5-fluorouridine(5′-DFUR). Capecitabine is marketed by Roche under the trade nameXeloda®.

Leucovorin (Folinic acid) is an adjuvant used in cancer therapy. It isused in synergistic combination with 5-FU to improve efficacy of thechemotherapeutic agent. Without being bound by theory, addition ofLeucovorin is believed to enhance efficacy of 5-FU by inhibitingthymidylate synthase. It has been used as an antidote to protect normalcells from high doses of the anticancer drug methotrexate and toincrease the antitumor effects of fluorouracil (5-FU) andtegafur-uracil. It is also known as citrovorum factor and Wellcovorin.This compound has the chemical designation of L-Glutamic acidN[4[[(2-amino-5-formyl1,4,5,6,7,8hexahydro4oxo6-pteridinyl)methyl]amino]b-enzoyl],calcium salt (1:1).

“Oxaliplatin” (Eloxatin) is a platinum-based chemotherapy drug in thesame family as cisplatin and carboplatin. It is typically administeredin combination with fluorouracil and leucovorin in a combination knownas FOLFOX for the treatment of colorectal cancer. Compared to cisplatin,the two amine groups are replaced by cyclohexyldiamine for improvedantitumour activity. The chlorine ligands are replaced by the oxalatobidentate derived from oxalic acid in order to improve water solubility.Equivalents to Oxaliplatin are known in the art and include, but are notlimited to cisplatin, carboplatin, aroplatin, lobaplatin, nedaplatin,and JM-216 (see McKeage et al. (1997) J. Clin. Oncol. 201:1232-1237 andin general, Chemotherapy for Gynecological Neoplasm, Curr. Therapy andNovel Approaches, in the Series Basic and Clinical Oncology, Angioli etal. Eds., 2004).

“FOLFOX” is an abbreviation for a type of combination therapy that isused to treat cancer. This therapy includes 5-FU, oxaliplatin andleucovorin. “FOLFIRI” is an abbreviation for a type of combinationtherapy that is used treat cancer and comprises, or alternativelyconsists essentially of, or yet further consists of 5-FU, leucovorin,and irinotecan. Information regarding these treatments are available onthe National Cancer Institute's web site, cancer.gov, last accessed onJan. 16, 2008.

Irinotecan (CPT-11) is sold under the trade name of Camptosar. It is asemi-synthetic analogue of the alkaloid camptothecin, which is activatedby hydrolysis to SN-38 and targets topoisomerase I. Chemical equivalentsare those that inhibit the interaction of topoisomerase I and DNA toform a catalytically active topoisomerase I-DNA complex. Chemicalequivalents inhibit cell cycle progression at G2-M phase resulting inthe disruption of cell proliferation.

The term “adjuvant” therapy refers to administration of a therapy orchemotherapeutic regimen to a patient after removal of a tumor bysurgery. Adjuvant therapy is typically given to minimize or prevent apossible cancer reoccurrence. Alternatively, “neoadjuvant” therapyrefers to administration of therapy or chemotherapeutic regimen beforesurgery, typically in an attempt to shrink the tumor prior to a surgicalprocedure to minimize the extent of tissue removed during the procedure.

The phrase “first line” or “second line” or “third line” etc., refers tothe order of treatment received by a patient. First line therapyregimens are treatments given first, whereas second or third linetherapy are given after the first line therapy or after the second linetherapy, respectively. The National Cancer Institute defines first linetherapy as “the first treatment for a disease or condition. In patientswith cancer, primary treatment can be surgery, chemotherapy, radiationtherapy, or a combination of these therapies. First line therapy is alsoreferred to those skilled in the art as primary therapy and primarytreatment.” See National Cancer Institute website as www.cancer.gov,last visited on May 1, 2008. Typically, a patient is given a subsequentchemotherapy regimen because the patient did not shown a positiveclinical or sub-clinical response to the first line therapy or the firstline therapy has stopped.

As used herein, the term “antifolate” intends a drug or biologic thatimpairs the function of folic acids, e.g., an antimetabolite agent thatinhibits the use of a metabolite, i.e. another chemical that is part ofnormal metabolism. In cancer treatment, antimetabolites interfere withDNA production, thus cell division and growth of the tumor. Non-limitingexamples of these agents are dihydrofolate reductase inhibitors, such asmethotrexate, Aminopterin, and Pemetrexed; thymidylate synthaseinhibitors, such as Raltitrexed or Pemetrexed; purine based, i.e. anadenosine deaminase inhibitor, such as Pentostatin, a thiopurine, suchas Thioguanine and Mercaptopurine, a halogenated/ribonucleotidereductase inhibitor, such as Cladribine, Clofarabine, Fludarabine, or aguanine/guanosine: thiopurine, such as Thioguanine; or Pyrimidine based,i.e. cytosine/cytidine: hypomethylating agent, such as Azacitidine andDecitabine, a DNA polymerase inhibitor, such as Cytarabine, aribonucleotide reductase inhibitor, such as Gemcitabine, or athymine/thymidine: thymidylate synthase inhibitor, such as aFluorouracil (5-FU).

In one aspect, the term “chemical equivalent” means the ability of thechemical to selectively interact with its target protein, DNA, RNA orfragment thereof as measured by the inactivation of the target protein,incorporation of the chemical into the DNA or RNA or other suitablemethods. Chemical equivalents include, but are not limited to, thoseagents with the same or similar biological activity and include, withoutlimitation a pharmaceutically acceptable salt or mixtures thereof thatinteract with and/or inactivate the same target protein, DNA, or RNA asthe reference chemical.

The terms “oligonucleotide” or “polynucleotide” or “portion,” or“segment” thereof refer to a stretch of polynucleotide residues which islong enough to use in PCR or various hybridization procedures toidentify or amplify identical or related parts of mRNA or DNA molecules.The polynucleotide compositions of this invention include RNA, cDNA,genomic DNA, synthetic forms, and mixed polymers, both sense andantisense strands, and may be chemically or biochemically modified ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those skilled in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

When a genetic marker, e.g., over expression of dUTPase, is used as abasis for selecting a patient for a treatment described herein, thegenetic marker is measured before and/or during treatment, and thevalues obtained are used by a clinician in assessing any of thefollowing: (a) probable or likely suitability of an individual toinitially receive treatment(s); (b) probable or likely unsuitability ofan individual to initially receive treatment(s); (c) responsiveness totreatment; (d) probable or likely suitability of an individual tocontinue to receive treatment(s); (e) probable or likely unsuitabilityof an individual to continue to receive treatment(s); (f) adjustingdosage; (g) predicting likelihood of clinical benefits; or (h) toxicity.As would be well understood by one in the art, measurement of thegenetic marker in a clinical setting is a clear indication that thisparameter was used as a basis for initiating, continuing, adjustingand/or ceasing administration of the treatments described herein.

“Cancer” is a known medically as a malignant neoplasm, is a broad groupof diseases involving unregulated cell growth. In cancer, cells divideand grow uncontrollably, forming malignant tumors, and invade nearbyparts of the body. Non-limiting examples include colon cancer,colorectal cancer, gastric cancer, esophogeal cancer, head and neckcancer, breast cancer, lung cancer, stomach cancer, liver cancer, gallbladder cancer, or pancreatic cancer or leukemia.

Compounds

In one aspect, provided herein are compounds of formula (I), (II), and(III):

or a tautomer thereof, or a pharmaceutically acceptable salt of eachthereof, wherein

is a uracil isostere or a halo uracil;

is uracil, halo uracil, or a uracil isostere;W is a bond or optionally substituted —CH₂—;W¹ is a bond, N, or an optionally substituted CH group;X is a bond, O, S, NR¹⁹, optionally substituted C₁-C₆ alkylene,optionally substituted C₂-C₆ alkenylene, or optionally substituted C₂-C₆alkynylene group, a divalent optionally substituted C₆-C₁₀ aromatichydrocarbon group, or a divalent optionally substituted saturated orunsaturated C₂-C₁₀ heterocyclic or optionally substituted C₁-C₁₀heteroaryl group;R¹⁹ is hydrogen, optionally substituted C₁-C₆ alkyl or optionallysubstituted C₃-C₈ cycloalkyl;Y is a bond or an optionally substituted C₁-C₁₀ alkylene which furtheroptionally has a cycloalkylidene structure on one carbon atom, or isoptionally substituted C₂-C₆ alkenylene, or optionally substituted C₂-C₆alkynylene group, or Y is —L¹⁰-B¹-L¹¹-;L¹⁰ and L¹¹ in dependently are optionally substituted C₁-C₆ alkylene,optionally substituted C₂-C₆ alkenylene, or optionally substituted C₂-C₆alkynylene group;B¹ is a divalent optionally substituted C₆-C₁₀ aromatic hydrocarbongroup, or a divalent optionally substituted saturated or unsaturatedC₂-C₁₀ heterocyclic or optionally substituted C₁-C₁₀ heteroaryl group;Z is —PO₂—NR³¹R³², —SO₂NR³¹R³², —NR³PO₂—R⁴, —NR³SO₂—R⁴, or R⁴ whereinR³¹ and R³² are the same or different and each represents a hydrogenatom, optionally substituted C₁-C₆ alkyl group optionally substitutedwith an aryl group, wherein the aryl group, together with the R³¹ orR³², may form a condensed bicyclic hydrocarbon, or R³¹ and R³² are takentogether with the adjacent nitrogen atom form an optionally substitutedC₂-C₁₀ heterocyclic group or an optionally substituted C₁-C₁₀ heteroarylgroup;Z¹ is —PO₂—NR³¹R³² or —(OR³)P(O)—R⁴ wherein R³¹ and R³² areindependently a hydrogen atom, optionally substituted C₁-C₆ alkyl groupoptionally substituted with an aryl group, wherein the aryl group,together with the R³¹ or R³², may form a condensed bicyclic hydrocarbon,or R³¹ and R³² taken together with the adjacent nitrogen atom form anoptionally substituted C₂-C₁₀ heterocyclic group or an optionallysubstituted C₁-C₁₀ heteroaryl group;R³ is hydrogen or optionally substituted C₁-C₆ alkyl; andR⁴ is optionally substituted C₆-C₁₀ aryl, an optionally substitutedC₂-C₁₀ heterocyclic group, or an optionally substituted C₁-C₁₀heteroaryl group.

In one embodiment, provided herein is a compound of formula (III):

wherein A is an uracil isostere selected from:

R¹⁰ is hydrogen, R¹², or —O—R¹²,R¹² is C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionallysubstituted with 1-3 hydroxy, fluoro, chloro, and amino substituent,R¹¹ is hydrogen, halo, R¹² or —O—R¹², wherein R¹² is defined as above,r is 1, 2, or 3,

L¹- is

wherein

Y¹ is CH₂, O, S, X¹⁰ is NH, NCO₂R²⁰, O, or CH₂,

R²⁰ is C₁-C₆ alkyl optionally substituted with 1-3 C₆-C₁₀ aryl groups,u is 0, 1, 2, 3, or 4,R^(z) is hydroxy or hydrogen,R^(w) is C₁-C₆ alkyl or hydrogen, andthe phenylene and the heteroarylene rings are optionally substituted,Z is phenyl or a 5 or 6 member heteroaryl substituted with an R⁶ and anR⁶⁰ groups, wherein the R⁶ and the R⁶⁰ are positioned 1,2 with respectto each other,R⁶ is hydrogen, optionally substituted C₁-C₆ alkoxy, or halo, and

R⁶⁰ is —OR⁷ or —NHR⁷R⁷⁰,

R⁷ is optionally substituted C₁-C₁₀ alkyl, optionally substituted C₂-C₆alkenyl, optionally substituted C₂-C₆ alkynyl, optionally substitutedC₃-C₈ cycloalkyl, optionally substituted C₃-C₁₀ heteroaryl, optionallysubstituted C₃-C₁₀ heterocyclyl, or optionally substituted phenyl, andR⁷⁰ is hydrogen or R⁷.

In another embodiment, the uracil isostere is an optionally substitutedcycloalkyl or optionally substituted heterocyclyl ring which ismonocyclic, bicyclic, tricyclic, or tetracyclic, wherein the ringcomprises a moiety selected from —C(═V)—NH—C(═V)—, —C(═V)—CH₂—C(═V)—.

In another embodiment, the uracil isostere is optionally substitutedmeta-dihaho phenyl or optionally substituted 1,3-dihalosubstitutedC₃-C₁₀ heteroaryl. In another embodiment, the uracil isostere isoptionally substituted meta-difluoro phenyl or meta-fluoro-halo phenyl.

In certain embodiments, the uracil isotere is halo uracil. In certainembodiments, the uracil isotere, particularly for formulas (I) and (II)are not halo uracil. As used herein, halo uracil refers to a halogenateduracil, a non limiting example of which includes 5-halo uracil.

In another embodiment, the uracil isostere is of formula:

wherein each V independently is O or S,each R¹ independently is hydrogen, C₁-C₆ alkyl optionally substitutedwith C₃-C₅ cycloalkyl, or C₃-C₅ cycloalkyl,each R² is independently —OH, —SH, —OR¹, —SR¹, or halo wherein R¹ isdefined as above,each Q¹ and Q² independently are —CH₂—, O, S or an oxidized formthereof, NH or an oxidized form thereof, or Q¹ and Q² together form a—CH═CH— moiety;provided that Q¹ and Q² are both not O, S or an oxidized form thereof,NH or an oxidized form thereof or a combination of each thereof;wherein each —CH═, —CH₂—, and —NH— is optionally substituted.

In another embodiment, R¹ independently is hydrogen or methyl. Inanother embodiment, each R¹ is hydrogen. In another embodiment, each R¹is methyl. In another embodiment, each V independently is O. In anotherembodiment, each V independently is S.

In another embodiment, each Q¹ independently is O. In anotherembodiment, each Q¹ independently is S. In another embodiment, each Q¹independently is optionally substituted —CH₂—. In another embodiment,each Q¹ independently is optionally substituted —NH—.

In another embodiment, each Q² independently is O. In anotherembodiment, each Q² independently is S. In another embodiment, each Q²independently is optionally substituted —CH₂—. In another embodiment,each Q¹ independently is optionally substituted —NH—.

In another embodiment, the uracil isostere is:

In some embodiments, the uracil isostere is:

In some embodiments, the uracil isostere is:

In some embodiments, the uracil isostere is:

In some embodiments, the uracil isostere is:

In another embodiment, —W—X—Y— is —CH₂—X—SO₂—NH—CH(R^(Y))—;—CH₂—X—SO₂—NH—C(R^(Y))₂—; or —CH₂—X—B—CH₂CR^(Z)R^(W)—,

X is optionally substituted C₁-C₆ alkylene wherein one of the methylenegroups within the alkylene chain is optionally replaced with an O or Satom, such that X is optionally substituted alkylene or optionallysubstituted heteroalkylene;B is a optionally substituted C₃-C₁₀ heteroaryl;R^(Y) an R^(w) are independently hydrogen or C₁-C₆ alkyl; andR^(z) is hydrogen or hydroxy.

In one embodiment, B is a 5 membered heteroaryl containing up to 3 or 4heteroatoms selected from nitrogen, sulfur and oxygen. In oneembodiment, B is:

In another embodiment, —W—X—Y— or L¹ is

wherein

X¹⁰ is NH, NCO₂R²⁰, O, or CH₂,

R²⁰ is C₁-C₆ alkyl optionally substituted with 1-3 C₆-C₁₀ aryl groups,u is 0, 1, 2, 3, or 4,

Y¹ is CH₂, O or S,

R^(z) is hydroxy or hydrogen,R^(w) is C₁-C₆ alkyl or hydrogen,the phenylene and the heteroarylene rings are optionally substituted.

In some embodiments, —W—X—Y— or L¹ is

In some embodiments, —W—X—Y— or L¹ is

In another embodiment, R⁴ is optionally substituted C₆-C₁₀ aryl. Inanother embodiment, R⁴ is optionally substituted C₂-C₁₀ heterocyclicgroup. In another embodiment, R⁴ is optionally substituted C₁-C₁₀heteroaryl group. In another embodiment, when Y is -L¹⁰-B¹-L¹¹-, Z isR⁴.

In some embodiments, Z is phenyl or a 5 or 6 membered heteroarylsubstituted with an R⁶ and an R⁶⁰ groups, wherein the R⁶ and the R⁶⁰ arepositioned 1,2 with respect to each other,

R⁶ is hydrogen, optionally substituted C₁-C₆ alkoxy, or halo, and

R⁶⁰ is —OR⁷ or —NHR⁷R⁷⁰,

R⁷ is optionally substituted C₁-C₆ alkyl, optionally substituted C₂-C₆alkenyl, optionally substituted C₂-C₆ alkynyl, optionally substitutedC₃-C₅ cycloalkyl, optionally substituted C₃-C₁₀ heteroaryl, optionallysubstituted C₃-C₁₀ heterocyclyl, or optionally substituted phenyl, andR⁷⁰ is hydrogen or R⁷.

In some embodiments, Z or R⁴ is selected from:

wherein each R⁶ and R⁷ independently are defined as in any aspect orembodiment above,each R⁶¹and R⁶² independently is N or CH, provided that at least one ofR⁶¹and R⁶² is N,each R⁶³ independently is NR⁷⁰, S, O, andeach R⁶⁴ independently is N or CH.

In some embodiments, provided herein is a compound of formula:

wherein L₁ is as defined above.

In some embodiments, provided herein is a compound of formula:

wherein L₁ is as defined above.

In another embodiment, Z is:

R⁶ is hydrogen, optionally substituted C₁-C₆ alkoxy, or halo, andR⁷ is optionally substituted C₁-C₆ alkyl, optionally substituted C₂-C₆alkenyl, optionally substituted C₂-C₆ alkynyl, optionally substitutedC₃-C₅ cycloalkyl, optionally substituted C₃-C₁₀heteroaryl, optionallysubstituted C₃-C₁₀ heterocyclyl, or optionally substituted phenyl.

In one embodiment, R⁶ is hydrogen. In one embodiment, R⁶ is halo. Inanother embodiment, R⁶ is fluoro. In one embodiment, R⁶ is C₁-C₆ alkoxy.In one embodiment, R⁶ is C₁-C₆ alkoxy substituted with 1-3 fluorogroups. In some embodiments, R⁶ is hydrogen, F, Cl, OMe, or OCF₃.

In one embodiment, R⁷ is C₁-C₆ alkyl substituted with a C₃-C₈cycloalkyl, C₂-C₁₀ heterocyclyl, or C₁-C₁₀ heteroaryl. In oneembodiment, R⁷ is

In one embodiment, R₇ is C₁-C₆ alkyl optionally substituted with a C₃-C₈cycloalkyl, 4-8 membered heterocyclyl, or R⁷ is C₁-C₆ alkyl substitutewith 1-3 fluoro atoms.

In another embodiment, R⁷ is:

wherein t is 1, 2, or 3. In another embodiment, t is 1. In anotherembodiment, t is 2. In another embodiment, t is 3.

In another embodiment, the cycloalkyl is cyclopropyl. In anotherembodiment, the cycloalkyl is cyclobutyl. In another embodiment, thecycloalkyl is cyclopentyl. In another embodiment, the cycloalkyl iscyclohexyl. In another embodiment, R⁷ is isobutyl. In anotherembodiment, R⁷ is neopentyl.

In another embodiment, the heterocyclyl is

In another embodiment, the heterocyclyl is:

In another embodiment, the heterocyclyl is:

In one embodiment, the compound is PCI10213 of formula:

In another embodiment, the compound is of formula:

wherein L₁ is as defined above.

In another embodiment, the compound is of formula:

wherein L₁ is as defined above.

In another embodiment, the compound is of formula:

wherein L₁ is as defined above.

In another embodiment, the compound is of formula:

wherein L₁ is as defined above.

In one embodiment, provided herein is a compound of formula:

wherein A is selected from:

X¹⁰ is NH, NCO₂R²⁰, O, or CH₂;

R²⁰ is C₁-C₆ alkyl optionally substituted with 1-3 C₆-C₁₀ aryl groups;u is 0, 1, 2, 3, or 4;R¹¹ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl whereineach alkyl, alkenyl, and alkynyl is optionally substituted with 1-3hydroxy, fluoro, chloro, and amino substituent;R₆₀ is C₁-C₆ alkyl andr is 1, 2, or 3.

In one embodiment, A is:

In another embodiment, A is selected from:

In another embodiment, X¹⁰ is CH₂ or NH. In another embodiment, t is 1.In another embodiment, t is 2. In another embodiment, t is 3.

In another embodiment, provided herein is a compound selected from:

and a diastereomer or an enantiomer thereof.

In another embodiment, provided here are the compounds:

d pharmaceutically acceptable salts thereof.

The compounds provided herein include individual, separated enantiomersand diastereomers, tautotomers, and pharmaceutically acceptable salts ofeach thereof, wherever applicable. In one aspect, the compounds areprovided as stereochemical pure, e.g., PCI 10586 and pharmaceuticallyacceptable salts thereof, as described herein. As used herein, the termstereochemically pure denotes a compound which has 80% or greater byweight of the indicated stereoisomer and 20% or less by weight of otherstereoisomers. In a further aspect, the compounds as described hereinhave 90% or greater by weight of the denoted stereoisomer and 10% orless by weight of other stereoisomers. In a yet further embodiment, thecompounds of this disclosure have 95% or greater by weight of thedenoted stereoisomer and 5% or less by weight of other stereoisomers. Ina still further embodiment, the compounds have 97% or greater by weightof the denoted stereoisomer and 3% or less by weight of otherstereoisomers.

Synthesis

The following general synthetic scheme is used to prepare the compoundsprovided herein. For example, compounds of formula I are synthesized asshown in the reaction scheme below.

In general, uracil, uracil isostere, or a halo uracil is treated with asuitable base such as butyl lithium in a solvent such as tetrahydrofuranor dimethylformamide. The A(−) anion can also be generated by halogenexchange of an A-halo bond with an alkyl lithium. It is then coupledwith compound B, wherein LG is a leaving group such as halogen, tosylateor mesylate to provide compounds of formula (1). In some embodiments,protection of an NH, OH, or such other group in uracil, uracil isostere,halo uracil, or the —W—X—Y—Z moiety is required. Compounds of formula(III) can also be synthesized in an analogous manner.

For example, compounds of formula II can be synthesized as schematicallyillustrated below:

Suitable conditions for the condensation reaction with the keto group,dehydration, formation of the Wittig reagent and the subsequent Wittingreaction, and the Schiff's base formation are well known to the skilledartisan.

Illustrative and non-limiting synthesis of other compounds containingother linkers, e.g., L1 or —W—X—Y—, are shown above.

A-ring substituted compounds provided here are synthesized as shownbelow and or following methods well known in the art in view of thepresent disclosure. See also, Journal of Heterocyclic Chemistry (2005)vol. 42, #2 p. 201-207, Journal of the American Chemical Society (2009)vol. 131, p. 8196-8210, Journal of Heterocyclic Chemistry (1994) vol.31, #2 p. 565-568, and Journal of Medicinal Chemistry (1994) vol. 37,#13 p. 2059-2070, each of which is incorporated herein by reference.

Additional —W—X—Y—Z moieties are disclosed in US 2011/0082163; US2012/0225838; Miyahara et al., J. Med. Chem. (2012) 55, 2970-2980;Miyakoshi et al., J. Med. Chem. (2012) 55, 2960-2969; Miyahara et al.,J. Med. Chem. (2012) 55 (11), pp 5483-5496; and Miyakoshi et al., J.Med. Chem. (2012) 55 (14), pp 6427-6437 (each of which are incorporatedherein by reference) and can be used with the A moieties disclosedherein.

These and other compounds provided herein are synthesized following artrecognized methods with the appropriate substitution of commerciallyavailable reagents as needed. For example, and without limitation,methods for synthesizing certain other compounds are described in US2011/0082163; US 2012/0225838; Miyahara et al., J. Med. Chem. (2012) 55,2970-2980; Miyakoshi et al., J. Med. Chem. (2012) 55, 2960-2969;Miyahara et al., J. Med. Chem. (2012) 55 (11), pp 5483-5496; andMiyakoshi et al., J. Med. Chem. (2012) 55 (14), pp 6427-6437 (eachsupra), which methods can be adapted by the skilled artisan upon readingthis disclosure and/or based on synthetic methods well known in the art,to prepare the compounds provided herein. Protection deprotectionmethods and protecting groups useful for such purposes are well known inthe art, for example in Greene's Protective Groups in Organic Synthesis,4^(th) Edition, Wiley, 2006, or a later edition of the book.

The compounds and the intermediates are separated from the reactionmixture, when desired, following art known methods such ascrystallization, chromatography, distillation, and the like. Thecompounds and the intermediates are characterized by art known methodssuch as thin layer chromatography, nuclear magnetic resonancespectroscopy, high performance liquid chromatography, and the like. Asdescribed in detail herein, a racemic mixture of the compound can beseparated to the diastereomers and tested and used diagnostically ortherapeutically as described herein. Thus, in one aspect, the compoundis provided as a stereochemically pure enantiomer, e.g., PCI 10586 orPCI 10585, as described herein.

Methods of testing and using the compounds provided herein are performedfollowing art recognized in vitro (cell free), ex vivo or in vivomethods. For example, and without limitation, methods for testing andusing certain other compounds are described in US 2011/0082163; US2012/0225838; Miyahara et al., J. Med. Chem. (2012) 55, 2970-2980;Miyakoshi et al., J. Med. Chem. (2012) 55, 2960-2969; Miyahara et al.,J. Med. Chem. (2012) 55 (11), pp 5483-5496; Miyakoshi et al., J. Med.Chem. (2012) 55 (14), pp 6427-6437 (each of which in incorporated byreference), which methods can be adapted by the skilled artisan uponreading this disclosure and/or based on methods well known in the art,to test and use the compounds provided herein.

Compositions

Compositions, including pharmaceutical compositions comprising thecompounds described herein can be manufactured by means of conventionalmixing, dissolving, granulating, dragee-making levigating, emulsifying,encapsulating, entrapping, or lyophilization processes. The compositionscan be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients, orauxiliaries which facilitate processing of the compounds provided hereininto preparations which can be used pharmaceutically.

The compounds of the technology can be administered by parenteral (e.g.,intramuscular, intraperitoneal, intravenous, ICV, intracisternalinjection or infusion, subcutaneous injection, or implant), oral, byinhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g.,urethral suppository) or topical routes of administration (e.g., gel,ointment, cream, aerosol, etc.) and can be formulated, alone ortogether, in suitable dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants, excipients,and vehicles appropriate for each route of administration.

In one embodiment, this technology relates to a composition comprising acompound as described herein and a carrier.

In another embodiment, this technology relates to a pharmaceuticalcomposition comprising a compound as described herein and apharmaceutically acceptable carrier.

In another embodiment, this technology relates to a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundas described herein and a pharmaceutically acceptable carrier.

The pharmaceutical compositions for the administration of the compoundscan be conveniently presented in dosage unit form and can be prepared byany of the methods well known in the art of pharmacy. The pharmaceuticalcompositions can be, for example, prepared by uniformly and intimatelybringing the compounds provided herein into association with a liquidcarrier, a finely divided solid carrier or both, and then, if necessary,shaping the product into the desired formulation. In the pharmaceuticalcomposition the compound provided herein is included in an amountsufficient to produce the desired therapeutic effect. For example,pharmaceutical compositions of the technology may take a form suitablefor virtually any mode of administration, including, for example,topical, ocular, oral, buccal, systemic, nasal, injection, infusion,transdermal, rectal, and vaginal, or a form suitable for administrationby inhalation or insufflation.

For topical administration, the compounds can be formulated assolutions, gels, ointments, creams, suspensions, etc., as is well-knownin the art.

Systemic formulations include those designed for administration byinjection (e.g., subcutaneous, intravenous, infusion, intramuscular,intrathecal, or intraperitoneal injection) as well as those designed fortransdermal, transmucosal, oral, or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions,or emulsions of the compounds provided herein in aqueous or oilyvehicles. The compositions may also contain formulating agents, such assuspending, stabilizing, and/or dispersing agents. The formulations forinjection can be presented in unit dosage form, e.g., in ampules or inmultidose containers, and may contain added preservatives.

Alternatively, the injectable formulation can be provided in powder formfor reconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, and dextrose solution, before use.To this end, the compounds provided herein can be dried by any art-knowntechnique, such as lyophilization, and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants are knownin the art.

For oral administration, the pharmaceutical compositions may take theform of, for example, lozenges, tablets, or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone,or hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose, or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). The tablets can be coated by methods well known in theart with, for example, sugars, films, or enteric coatings.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions, and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents, and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain thecompounds provided herein in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients can be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents (e.g., corn starch oralginic acid); binding agents (e.g. starch, gelatin, or acacia); andlubricating agents (e.g., magnesium stearate, stearic acid, or talc).The tablets can be left uncoated or they can be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate can be employed. They may also becoated by the techniques well known to the skilled artisan. Thepharmaceutical compositions of the technology may also be in the form ofoil-in-water emulsions.

Liquid preparations for oral administration may take the form of, forexample, elixirs, solutions, syrups, or suspensions, or they can bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives, orhydrogenated edible fats); emulsifying agents (e.g., lecithin, oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol, Cremophore™, or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, preservatives, flavoring,coloring, and sweetening agents as appropriate.

Use of Compounds for Preparing Medicaments

The compounds and compositions of the present invention are also usefulin the preparation of medicaments to treat a variety of pathologies asdescribed herein. The methods and techniques for preparing medicamentsof a composition are known in the art.

For the purpose of illustration only, pharmaceutical formulations androutes of delivery are detailed herein.

Thus, one of skill in the art would readily appreciate that any one ormore of the compositions described above, including the many specificembodiments, can be used by applying standard pharmaceuticalmanufacturing procedures to prepare medicaments to treat the manydisorders described herein. Such medicaments can be delivered to thesubject by using delivery methods known in the pharmaceutical arts.

Methods and Therapies

The compositions and compounds as disclosed herein are useful in methodsof inhibiting dUTPase or enhancing the efficacy of a dUTPase-directedtherapy, or yet further, reversing resistance to dUTPase therapies. Themethods comprise, or alternatively consist essentially of, or yetfurther consist of, contacting the dUTPase with an effective amount ofthe compound or composition as disclosed herein. In one embodiment, themethods further comprise, or alternatively consist essentially of, oryet further consist of, contacting the dUTPase with an effective amountof a dUTPase-directed therapy. In one aspect, the contacting of thedUTPase-directed therapy is prior to, concurrent or subsequent tocontacting with the compound or composition of this disclosure.

One of skill in the art can also determine if the compound orcombination inhibits dUTPase in vitro by contacting the compound orcombination with purified or recombinant dUTPase in a cell free system.The purified or recombinant dUTPase and can be from any species, e.g.,simian, canine, bovine, ovine, rat, mouse or human. In one aspect, thedUTPase is DUT-N or DUT-M. Isolation, characterization and expression ofdUTPase isoforms are disclosed in U.S. Pat. No. 5,962,246 and known inthe art.

The contacting can be performed cell-free in vitro or ex vivo with acell or in a cell culture. When performed in vitro or ex vivo, thecompounds, compositions or agents can be directly added to the enzymesolution or added to the cell culture medium. When practiced in vitro orex vivo, the method can be used to screen for novel combinationtherapies, formulations or treatment regimens, prior to administrationto administration to an animal or a human patient. Methods to quantifyinhibition are known in the art, see, U.S. Patent Publ. Nos.2010/0075924 and 2011/0212467 and U.S. Pat. No. 7,601,702. For example,a fixed dose of a dUTPase directed therapy (e.g., 5-FU or Pemetrexed)can be added to the system and varying amounts of the compound can besubsequently added to system. Alternatively, a fixed dose of a compoundof this invention can be added to the system and varying amounts of thedUTPase directed therapy (e.g., 5-FU or Pemetrexed) compound can besubsequently added to system.

In one aspect, the contacting is ex vivo and the cell or tissue to becontacted over expresses dUTPase. These cells can be isolated from apatient prior to administration to the patient or can be purchased froma depository such as the American Type Culture Collection (ATCC).Non-limiting examples of animal (e.g., canine, an equine, a bovine, afeline, an ovine, a mouse, a rat or a simian) and human cells that areknown to over express dUTPase include, without limitation cancer cells,e.g. colon cancer, colorectal cancer, gastric cancer, head and neckcancer, breast cancer, stomach cancer or lung cancer. The cancer can bemetastatic or non-metastatic. Methods to quantify inhibition are knownin the art, see, U.S. Patent Publ. Nos. 2010/0075924 and 2011/0212467and U.S. Pat. No. 7,601,702 and Wilson et al. (2012) Mol. Cancer Ther.11:616-628.

When practiced in vivo in a patient such as an animal or human, thecompounds, compositions or agents are administered in an effectiveamount by a suitable route of administration, as determined by atreating physician taking into account the patient, disease and otherfactors. When practiced in a non-human animal, e.g., an appropriatemouse model, the method can be used to screen for novel combinationtherapies, formulations or treatment regimens, prior to administrationto a human patient.

This disclosure also provides methods of treating a disease whosetreatment is impeded by the expression of dUTPase, comprising, oralternatively consisting essentially of, or yet further consisting of,administering to a patient in need of such treatment an effective amountof the compound or composition of this disclosure, thereby treating thedisease. In one aspect, the method further comprises isolating a cell ortissue sample from the patient and screening for the expression level ofdUTPase, wherein over expression of dUTPase in the sample as compared toa control sample serves as a basis for selecting the patient as suitablefor the method and therapies. Methods to quantify dUTPase are known inthe art. Effective amounts will vary with the patient, the disease andthe general health of the patient and are determined by the treatingphysician. Methods to quantify inhibition are known in the art, see,U.S. Patent Publ. Nos. 2010/0075924 and 2011/0212467 and U.S. Pat. No.7,601,702 and Wilson et al. (2012) Mol. Cancer Ther. 11:616-628. If thepatient sample shows over expression of dUTPase, the therapy isadministered to the patient. If the patient sample does not show overexpression, an alternate therapy is chosen. The screen can be repeatedthroughout therapy as a means to monitor the therapy and/or dosageregimen.

To practice this method, the sample is a patient sample containing thetumor tissue, normal tissue adjacent to said tumor, normal tissue distalto said tumor or peripheral blood lymphocytes. In a further aspect, thepatient or patient population to be treated also is treatment naïve.

In one aspect, the method also requires isolating a sample containingthe genetic material to be tested; however, it is conceivable that oneof skill in the art will be able to analyze and identify genetic markersin situ at some point in the future. Accordingly, in one aspect, theinventions of this application are not to be limited to requiringisolation of the genetic material prior to analysis.

These methods also are not limited by the technique that is used toidentify the expression level or in aspects where expression has beenlinked to a polymorphism, the polymorphism of interest. Suitable methodsinclude but are not limited to the use of hybridization probes,antibodies, primers for PCR analysis, and gene chips, slides andsoftware for high throughput analysis. Additional genetic markers can beassayed and used as negative controls.

In one aspect, the subject or patient is an animal or a human patient.Non-limiting examples of animals include a feline, a canine, a bovine,an equine, an ovine, a mouse, a rat or a simian.

Diseases in which treatment is impeded by the expression of dUTPaseinclude, without limitation, cancer, viral infection, bacterialinfection or an autoimmune disorder. For example, in rheumatoidarthritis, inflammatory bowel disease or other autoimmune disorders, adUTPase inhibitor can be used in combination with an antifolate orfluoropyrimidine or other thymidylate synthase and dihydrofolatereductase inhibitors; parasitic, viral or bacterial infections can betreated similarly employing a combination therapy including a dUTPaseinhibitor. Non-limiting examples of cancer include, colon cancer,colorectal cancer, gastric cancer, head and neck cancer, breast cancer,stomach cancer, lung cancer or a leukemia. The cancer can be metastaticor non-metastatic.

In one aspect, the compound or composition is administered as one ormore of: a first line therapy or alternatively, a second line therapy, athird line therapy, or a fourth or subsequent line therapy toadministration of a dUPTase-directed therapy. Non-limiting examples ofdUTPase-directed therapies include an antimetabolite or afluoropyrmidine therapy or a 5-FU based adjuvant therapy or anequivalent or each thereof, such as 5-FU, tegafur, gimeracil, oteracilpotassium, capcitabine, 5-fluoro-2′-deoxyuridine, methotrexate, orpemetrexed or an equivalent of each thereof.

Certain compounds provided herein demonstrated substantial, such as,20-100% DUTPase inhibitory effect, e.g., an ability to inhibit dUTPaseunder conditions described herein below, and/or known to the skilledartisan, compared, for example, a compound provided herein:

In one embodiment, certain therapeutic methods provided herein excludethe use of the compounds PCI 10898, 10897, 10928, and 10929.

Kits

The compounds and compositions, as described herein, can be provided inkits. The kits can further contain additional dUTPase inhibitors andoptionally, instructions for use. In a further aspect, the kit containsreagents and instructions to perform the screen to identify patientsmore likely to respond to the therapy as described above.

Screening Assays

This invention also provides screening assays to identify potentialtherapeutic agents of known and new compounds and combinations. Forexample, one of skill in the art can also determine if the compound orcombination inhibits dUTPase in vitro by contacting the compound orcombination with purified or recombinant dUTPase in a cell free system.The purified or recombinant dUTPase and can be from any species, e.g.,simian, canine, bovine, ovine, rat, mouse or human. In one aspect, thedUTPase is DUT-N or DUT-M. Isolation, characterization and expression ofdUTPase isoforms are disclosed in U.S. Pat. No. 5,962,246 and known inthe art.

The contacting can be performed cell-free in vitro or ex vivo with acell or in a cell culture. When performed in vitro or ex vivo, thecompounds, compositions or agents can be directly added to the enzymesolution or added to the cell culture medium. When practiced in vitro orex vivo, the method can be used to screen for novel combinationtherapies, formulations or treatment regimens, prior to administrationto administration to an animal or a human patient. Methods to quantifyinhibition are known in the art, see, U.S. Patent Publ. Nos.2010/0075924 and 2011/0212467 and U.S. Pat. No. 7,601,702. For example,a fixed dose of a dUTPase directed therapy (e.g., 5-FU or Pemetrexed)can be added to the system and varying amounts of the compound can besubsequently added to system. Alternatively, a fixed dose of a compoundof this invention can be added to the system and varying amounts of thedUTPase directed therapy (e.g., 5-FU or Pemetrexed) compound can besubsequently added to system.

In another aspect, the assay requires contacting a first samplecomprising suitable cells or tissue (“control sample”) with an effectiveamount of a composition of this invention and optionally a dUTPaseinhibitor, and contacting a second sample of the suitable cells ortissue (“test sample”) with the agent to be assayed and optionally adUTPase inhibitor. In one aspect, the cell or tissue over expressdUTPase. The inhibition of growth of the first and second cell samplesare determined. If the inhibition of growth of the second sample issubstantially the same or greater than the first sample, then the agentis a potential drug for therapy. In one aspect, substantially the sameor greater inhibition of growth of the cells is a difference of lessthan about 1%, or alternatively less than about 5% or alternatively lessthan about 10%, or alternatively greater than about 10%, oralternatively greater than about 20%, or alternatively greater thanabout 50%, or alternatively greater than about 90%. The contacting canbe in vitro or in vivo. Means for determining the inhibition of growthof the cells are well known in the art.

In a further aspect, the test agent is contacted with a third sample ofcells or tissue comprising normal counterpart cells or tissue to thecontrol (or alternatively cells that do not over express dUTPase) andtest samples and selecting agents that treat the second sample of cellsor tissue but does not adversely effect the third sample. For thepurpose of the assays described herein, a suitable cell or tissue isdescribed herein such as cancer or other diseases as described herein.Examples of such include, but are not limited to cancer cell or tissueobtained by biopsy, blood, breast cells, colon cells.

Efficacy of the test composition is determined using methods known inthe art which include, but are not limited to cell viability assays orapoptosis evaluation.

In yet a further aspect, the assay requires at least two cell types, thefirst being a suitable control cell.

The assays also are useful to predict whether a subject will be suitablytreated by this invention by delivering a composition to a samplecontaining the cell to be treated and assaying for treatment which willvary with the pathology or for screening for new drugs and combinations.In one aspect, the cell or tissue is obtained from the subject orpatient by biopsy. Applicants provide kits for determining whether apathological cell or a patient will be suitably treated by this therapyby providing at least one composition of this invention and instructionsfor use.

The test cells can be grown in small multi-well plates and is used todetect the biological activity of test compounds. For the purposes ofthis invention, the successful candidate drug will block the growth orkill the pathogen but leave the control cell type unharmed.

The following examples are included to demonstrate some embodiments ofthe disclosure. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 Synthesis of PCI 10213

Piperidine-2,6-dione was treated with a suitable base such as lithiumhexamethyldisilazide, lithiumdiisopropylamide (LDA), orLDA/hexamethylphosphoramide (HMPT) in tetrahydrofuran as a solvent. Itwas then coupled with the bromide or chloride as shown in step 2 above,followed by acid hydrolysis to remove the sulfonamide protecting group,to provide PCI 10213.

Other compounds of formula (I) and (III) were prepared in an analogousmanner. In some cases, protection of the “NH” group on thepiperidine-2,4-dione is required.

Example 2 Synthesis of PCI 10214

Thiazolidine-2,4-dione was treated with a suitable base such as n-butyllithium, secondary butyllithium, or LDA/HMPT in a solvent such astetrahydrofuran or dimethylformamide. It was then coupled with thebromide or chloride as shown in step 2 above, followed by acidhydrolysis to remove the sulfonamide protecting group, to provide PCI10214.

Other compounds of formula (I) and (III) can be and were prepared in ananalogous manner. In some cases, protection of the “NH” group on thethiazolidine-2,4-dione is required.

Example 3 Preparation of Stereochemically Pure Compounds

The disclosed compounds exist as two diastereomers differing at only onesingle stereo center. This example demonstrates a separation protocol.The stereochemical pure compounds were prepared and then tested todetermine if the biological activity is attributed to one or bothstereoisomers.

Separation of the diastereomers was performed by preparative chiral highperformance liquid chromatography (HPLC) employing a 250×30 mm CHIRALPAKIA (5 μm) column, heptane/iso-propanol (70/30) with a flow-rate of 42.5mL/min and UV detection (λ=270 nm at 25° C.). Analytical chiral HPLC wasperformed employing a 250×4.6 mm CHIRALPAK IA (5 μm) column,heptane/iso-propanol/diethylamine (70/30/0.1) with a flow rate of 1mL/min and UV detection (1=230 nm at 25° C.).

PCI 10213 exists as a mixture of diasteromers differing at the chiralcarbon shown in Example 1. PCI 10213 was separated by preparative chiralHPLC under the above specified conditions to provide enantiomers PCI10586 and PCI 10585 that were in >99% enantiomeric excess and >95%purity. FIGS. 9 and 10 show the chiral HPLC chromatograms of PCI 10586and PCI 10585 with retention times (R_(t)) of 28.4 and 22.13 mins,respectively.

Example 4 Key Intermediate I(S)-1-azido-2-(3-(cyclopropylmethoxy)-4-fluorophenyl)butan-2-ol

Key intermediate I was prepared according to the literature data (J.Med. Chem. 2012, 55, 6427).

General Procedure A Alkylation with LiHMDS

At −40° C., a solution of lithium bis(trimethylsilyl)amide 1 M intetrahydrofuran (38.9 mmol, 38.9 mL, 2.2 eq) was added dropwise to asolution of glutarimide (2.0 g, 17.7 mmol, 1.0 eq) in tetrahydrofuran(30 mL). The iodoalkane (53.1 mmol, 3.0 eq) was immediately added. After15 minutes at −40° C., the mixture was allowed to warm up and themixture was stirred at room temperature for 18 hours. The reaction wasquenched with a saturated solution of ammonium chloride (10 mL) and theaqueous phase was extracted with methylene chloride (3×20 mL). Thecombined organic phases were dried over magnesium sulfate, filtered andevaporated under reduced pressure. The residue was purified by flashchromatography using cyclohexane and ethyl acetate (100/0 to 0/100) toafford the expected compound.

General Procedure B Alkylation with LDA

At 0° C., a solution of lithium diisopropylamide 2 M intetrahydrofuran/heptane/ethylbenzene (38.9 mmol, 19.5 mL, 2.2 eq) wasadded dropwise to a solution of glutarimide (2.0 g, 17.7 mmol, 1.0 eq)in tetrahydrofuran (30 mL). The iodoalkane (53.1 mmol, 3.0 eq) wasimmediately added. After 15 minutes at 0° C., the mixture was allowed towarm up and then stirred at room temperature for 18 hours. The reactionwas quenched with water (10 mL) and the aqueous phase was extracted withmethylene chloride (3×20 mL). The combined organic phases were driedover magnesium sulfate, filtered and evaporated under reduced pressure.The residue was purified by flash chromatography using cyclohexane andethyl acetate (100/0 to 0/100) to afford the expected compound.

General Procedure C Reductive Amination

To a solution of the amino compound (HCl Salt) (1.0 eq) in methanol (10mL) was added a 7 N solution of ammonia in methanol (3.0 eq). Themixture was stirred at room temperature during 15 minutes and aceticacid was added until pH=5. The aldehyde (1.0 eq) and sodiumcyanoborohydride (3.0 eq) were added and the mixture was stirred at roomtemperature for 18 hours. The reaction mixture was carefully quenchedwith a saturated solution of sodium hydrogenocarbonate (10 mL). Theaqueous phase was extracted with ethyl acetate (3×15 mL). The combinedorganic phases were dried over magnesium sulfate, filtered andevaporated under reduced pressure. The residue was purified by flashchromatography using cyclohexane and ethyl acetate (100/0 to 0/100) toafford the expected compound.

General Procedure D “Click Chemistry”

To a solution of the alkynyl compound (1.0 eq) and Key Intermediate 1(1.0 eq) in dioxane (10 mL) degazed with argon was addedchloro(1,5-cyclooctadiene)(pentamethylcyclopentadienyl)ruthenium II (0.1eq). The reaction mixture was stirred at 80° C. for 3 hours. Aftercooling down, the reaction mixture was evaporated under vacuum and theresidue was absorbed on silica gel to be purified by flashchromatography using cyclohexane and ethyl acetate (100/0 to 0/100) toafford the expected compound.

Example 53-(4-(3-[(S)-2-(3-Cyclopropylmethoxy-4-fluorophenyl)-2-hydroxybutyl]-3H-[1,2,3]triazol-4-yl)-butyl)-piperidine-2,6-dione

Step 1

3-hex-5-ynyl-piperidine-2,6-dione was prepared according to GeneralProcedure A using glutarimide (2.0 g, 17.7 mmol) and 6-iodo-1-hexyne(5.6 mL, 42.4 mmol). The expected compound was isolated as orange oilthat solidified during storage with 17% yield (570 mg).

Step 2

The title compound was prepared according to General Procedure D, using3-hex-5-ynyl-piperidine-2,6-dione prepared in step 1 (173 mg, 0.9 mmol)and Key Intermediate I (250 mg, 0.9 mmol). The expected compound wasisolated as beige foam with 69% yield (291 mg).

¹H NMR (CDCl₃): 7.83 (broad s, 1H), 7.37 (s, 1H), 6.99 (ddd, J=1.5, 8.5and 12.4 Hz, 1H), 6.89 (dd, J=2.2 and 8.2 Hz, 1H), 7.76 (m, 1H), 4.45(d, J=14.0 Hz, 1H), 4.34 (d, J=14.0 Hz, 1H), 3.78 (d, J=7.0 Hz, 2H),2.72 (m, 1H), 2.56 (m, 1H), 2.36 (m, 3H), 2.21-1.70 (m, 6H), 1.53 (m,3H), 1.36 (m, 2H), 1.22 (m, 1H), 0.83 (t, J=7.3 Hz, 3H), 0.62 (m, 2H),0.32 (m, 2H)

Example 63-(5-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-pentyl)-piperidine-2,6-dione

Step 1

3-hept-6-ynyl-piperidine-2,6-dione was prepared according to GeneralProcedure A using glutarimide (2.0 g, 17.7 mmol) and 7-iodo-hept-1-yne(9.4 g, 42.5 mmol). The expected compound was isolated as beige powderwith 29% yield (1.07 g).

Step 2

The title compound was prepared according to General Procedure D, using3-hept-6-ynyl-piperidine-2,6-dione prepared in step 1 (185 mg, 0.9 mmol)and Key Intermediate I (250 mg, 0.9 mmol). The expected compound wasisolated as solidified oil with 67% yield (290 mg).

¹H NMR (CDCl₃): 7.80 (broad s, 1H), 7.36 (s, 1H), 6.99 (dd, J=8.5 and10.8 Hz, 1H), 6.90 (m, 1H), 6.77 (m, 1H), 4.45 (d, J=14.0 Hz, 1H), 4.34(dd, J=1.5 and 14.0 Hz, 1H), 3.78 (d, J=7.0 Hz, 2H), 2.72 (dt, J=4.8 and17.7 Hz, 1H), 2.55 (m, 1H), 2.42 (m, 1H), 2.27 (m, 2H), 2.12-1.69 (m,6H), 1.61-1.19 (m, 8H), 0.82 (t, J=7.3 Hz, 3H), 0.62 (m, 2H), 0.33 (m,2H)

Example 73-(3-(3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl)-propyl)-piperidine-2,6-dione

Step 1

3-pent-4-ynyl-piperidine-2,6-dione was prepared according to GeneralProcedure A using glutarimide (1.0 g, 8.8 mmol) and 5-iodo-pent-1-yne(5.0 g, 25.6 mmol). The expected compound was isolated as white powderwith 10% yield (152 mg).

Step 2

The title compound was prepared according to General Procedure D, using3-pent-4-ynyl-piperidine-2,6-dione prepared in step 1 (150 mg, 0.8 mmol)and Key Intermediate I (234 mg, 0.8 mmol). The expected compound wasisolated as white powder with 64% yield (246 mg) after purification andlyophilization.

¹H NMR (DMSO): 10.58 (s, 1H), 7.39 (s, 1H), 7.06 (dd, J=8.5 and 11.3 Hz,1H), 6.93 (dd, J=1.9 and 8.4 Hz, 1H), 6.82 (m, 1H), 5.28 (s, 1H), 4.40(s, 2H), 3.76 (d, J=7.0 Hz, 2H), 2.60-2.20 (m, 5H), 1.92 (m, 2H), 1.75(m, 2H), 1.51 (m, 3H), 1.35 (m, 1H), 1.16 (m, 1H), 0.66 (t, J=7.2 Hz,3H), 0.53 (m, 2H), 0.29 (m, 2H)

Example 83-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-butylamino)-piperidine-2,6-dione

Step 1

3-hex-5-ynylamino-piperidine-2,6-dione was prepared according to GeneralProcedure C using 3-aminopiperidine-2,6-dione hydrochloride (500 mg, 3.0mmol) and hex-5-ynal (292 mg, 3.0 mmol) prepared from hex-5-yn-1-olaccording to the procedure described in the literature (US2011/306551).Before addition of the solution of sodium hydrogenocarbonate (10 mL),the reaction mixture was concentrated. The expected compound wasisolated with 32% yield (203 mg).

Step 2

To a solution of 3-hex-5-ynylamino-piperidine-2,6-dione prepared in step1 (188 mg, 0.9 mmol, 1.0 eq) in acetonitrile (15 mL) were addeddi-tert-butyl dicarbonate (433 mg, 1.98 mmol, 2.2 eq) and4-dimethylaminopyridine (11 mg, 0.09 mmol, 0.1 eq). The mixture wasstirred at room temperature during 18 hours. The reaction was quenchedwith a saturated solution of sodium hydrogenocarbonate (10 mL) andextracted with ethyl acetate (3×15 mL). The combined organic phases weredried over magnesium sulfate, filtered and evaporated under reducedpressure. The residue was purified by flash chromatography usingcyclohexane and ethyl acetate (100/0 to 60/40) to afford(2,6-dioxo-piperidin-3-yl)-hex-5-ynyl-carbamic acid tert-butyl esterwith 50% yield (139 mg).

Step 3

(4-{3-[(S)-2-(3-cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-butyl)-(2,6-dioxo-piperidin-3-yl)-carbamicacid tert-butyl ester was prepared according to General Procedure D,using (2,6-dioxo-piperidin-3-yl)-hex-5-ynyl-carbamic acid tert-butylester prepared in step 2 (125 mg, 0.4 mmol) and Key Intermediate I (113mg, 0.4 mmol). The expected compound was obtained as beige foam with 68%yield (160 mg).

Step 4

To a solution of(4-{3-[(S)-2-(3-cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-butyl)-(2,6-dioxo-piperidin-3-yl)-carbamicacid tert-butyl ester prepared in step 3 (160 mg, 0.3 mmol, 1.0 eq) inmethylene chloride (10 mL) was added a 1 M solution of hydrochloride indiethyl ether (10 mL). After stirring at room temperature during 3hours, the mixture was concentrated and a saturated solution of sodiumhydrogenocarbonate (15 mL) was added. The aqueous phase was extractedwith ethyl acetate (3×15 mL). The combined organic phases were driedover magnesium sulfate, filtered and evaporated under reduced pressure.The residue was purified by flash chromatography using ethyl acetate andmethanol (100/0 to 80/20) and lyophilized to afford the expectedcompound as white solid with 54% yield (71 mg).

¹H NMR (DMSO): 10.66 (s, 1H), 7.36 (s, 1H), 7.06 (dd, J=8.5 and 11.3 Hz,1H), 6.92 (dd, J=2.1 and 8.4 Hz, 1H), 6.82 (m, 1H), 5.27 (s, 1H), 4.40(s, 2H), 3.76 (d, J=7.0 Hz, 2H), 3.26 (m, 1H), 2.65-2.40 (m, 3H), 2.25(m, 4H), 1.98 (m, 2H), 1.74 (m, 2H), 1.40 (m, 4H), 1.17 (m, 1H), 0.66(t, J=7.2 Hz, 3H), 0.55 (m, 2H), 0.30 (m, 2H)

Example 93-(3-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-propylamino)-piperidine-2,6-dione

Step 1

3-pent-4-ynylamino-piperidine-2,6-dione was prepared according toGeneral Procedure C using 3-aminopiperidine-2,6-dione hydrochloride (800mg, 4.9 mmol) and pent-4-ynal (1.5 g, 18.8 mmol) prepared frompent-5-yn-1-ol according to the procedure described in the literature(US2011/306551). Before addition of the solution of sodiumhydrogenocarbonate (10 mL), the reaction mixture was evaporated. Theexpected compound was isolated with 21% yield (200 mg).

Step 2

To a solution of 3-pent-4-ynylamino-piperidine-2,6-dione prepared instep 1 (190 mg, 1.0 mmol, 1.0 eq) in acetonitrile (15 mL) were addeddi-tert-butyl dicarbonate (470 mg, 2.2 mmol, 2.2 eq) and4-dimethylaminopyridine (12 mg, 0.1 mmol, 0.1 eq). The mixture wasstirred at room temperature for 18 hours. The reaction was quenched witha saturated solution of sodium hydrogenocarbonate (10 mL) and extractedwith ethyl acetate (3×15 mL). The combined organic phases were driedover magnesium sulfate, filtered and evaporated under reduced pressure.The residue was purified by flash chromatography using cyclohexane andethyl acetate (100/0 to 60/40) to afford(2,6-dioxo-piperidin-3-yl)-pent-4-ynyl-carbamic acid tert-butyl esterwith 60% yield (180 mg).

Step 3

(3-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-propyl)-(2,6-dioxo-piperidin-3-yl)-carbamicacid tert-butyl ester was prepared according to General Procedure D,using (2,6-dioxo-piperidin-3-yl)-pent-4-ynyl-carbamic acid tert-butylester prepared in step 2 (180 mg, 0.6 mmol, 1.0 eq) and Key IntermediateI (171 mg, 0.6 mmol, 1.0 eq). The compound was obtained as beige foamwith 49% yield (170 mg).

Step 4

To a solution of(3-{3-[(S)-2-(3-cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-propyl)-(2,6-dioxo-piperidin-3-yl)-carbamicacid tert-butyl ester prepared in step 3 (170 mg, 0.3 mmol, 1.0 eq) inmethylene chloride (10 mL) was added a 1 M solution of hydrochloride indiethyl ether (10 mL). After stirring at room temperature during 3hours, the mixture was concentrated and a saturated solution of sodiumhydrogenocarbonate (15 mL) was added. The aqueous phase was extractedwith ethyl acetate (3×15 mL). The combined organic phases were driedover magnesium sulfate, filtered and evaporated under reduced pressure.The residue was purified by flash chromatography using ethyl acetate andmethanol (100/0 to 90/10) and lyophilized to afford the title compoundwith 88% yield as light blue solid (125 mg).

¹H NMR (DMSO): 10.66 (s, 1H), 7.38 (s, 1H), 7.06 (dd, J=8.4 and 11.0 Hz,1H), 6.93 (d, J=8.4 Hz, 1H), 6.83 (m, 1H), 5.27 (s, 1H), 4.41 (s, 2H),3.77 (d, J=7.0 Hz, 2H), 2.25 (m, 1H), 2.57 (m, 3H), 2.36 (m, 3H), 2.21(m, 1H), 1.96 (m, 2H), 1.82-1.50 (m, 4H), 1.16 (m, 1H), 0.66 (t, J=7.2Hz, 3H), 0.55 (m, 2H), 0.29 (m, 2H)

Example 103-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluorophenyl)-2-hydroxybutyl]-3H-[1,2,3]triazol-4-yl}-butylamino)-3-methyl-piperidine-2,6-dione

Step 1

3-hex-5-ynylamino-3-methyl-piperidine-2,6-dione was prepared accordingto General Procedure C using 3-amino-3-methyl-piperidine-2,6-dionehydrochloride monohydrate prepared according the procedure described inthe literature (WO2006/081251) (600 mg, 3.0 mmol) and hex-5-ynal (440mg, 3.0 mmol) prepared from hex-5-yn-1-ol according to the proceduredescribed in the literature (US2011/306551). The expected compound wasisolated with 41% yield (280 mg).

Step 2

The title compound was prepared according to General Procedure D, using3-hex-5-ynylamino-3-methyl-piperidine-2,6-dione prepared in step 1 (100mg, 0.4 mmol) and Key Intermediate I (126 mg, 0.4 mmol). The expectedcompound was obtained as white powder after purification andlyophilization with 29% yield (65 mg).

¹H NMR (DMSO): 10.56 (s, 1H), 7.37 (s, 1H), 7.08 (dd, J=8.5 and 11.3 Hz,1H), 6.93 (dd, J=1.9 and 8.5 Hz, 1H), 6.85 (m, 1H), 5.29 (s, 1H), 4.41(s, 2H), 3.78 (d, J=7.0 Hz, 2H), 2.63 (m, 1H), 2.34 (m, 5H), 1.99 (m,3H), 1.76 (m, 2H), 1.43 (m, 2H), 1.31 (m, 2H), 1.18 (m, 4H), 0.68 (t,J=7.2 Hz, 3H), 0.56 (m, 2H), 0.31 (m, 2H)

Example 113-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one

Step 1

3-hex-5-ynyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one was preparedaccording to General Procedure B using3,4-dihydro-1H-[1,8]naphthyridin-2-one (300 mg, 2.0 mmol) and6-iodo-1-hexyne (790 μL, 6.0 mmol). The expected compound was isolatedas yellow powder with 13% yield.

Step 2

The title compound was prepared according to General Procedure D, using3-hex-5-ynyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one prepared in step 1(60 mg, 0.3 mmol) and Key Intermediate I (73 mg, 0.3 mmol). The expectedcompound was isolated as white powder after flash chromatography andlyophilization with 55% yield (73 mg).

¹H NMR (CDCl₃): 8.80 (broad s, 1H), 8.18 (d, J=4.3 Hz, 1H), 7.56 (d,J=7.3 Hz, 1H), 7.33 (s, 1H), 6.99 (m, 2H), 6.89 (m, 1H), 6.77 (m, 1H),4.43 (dd, J=1.7 and 14.0 Hz, 1H), 4.33 (d, J=14.0 Hz, 2H), 3.77 (dd,J=1.9 and 7.0 Hz, 2H), 3.03 (dd, J=6.0 and 16.0 Hz, 1H), 2.74 (m, 1H),2.57 (m, 1H), 2.28 (m, 2H), 2.00 (m, 1H), 1.84 (m, 2H), 1.60-1.30 (m,5H), 1.21 (m, 1H), 0.83 (t, J=7.4 Hz, 3H), 0.61 (m, 2H), 0.31 (m, 2H)

Example 123-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-butyl)-8-methoxy-3,4-dihydro-1H-quinolin-2-one

Step 1

To a solution of 2-chloro-8-methoxy-quinoline (2.1 g, 10.7 mmol, 1.0 eq)in acetic acid (15 mL) was added water (5 mL). The mixture was stirredat 100° C. during 18 hours. After cooling down, the solvent wasevaporated. Water (30 mL) and a 25% solution of ammonium hydroxide (20mL) were added. The aqueous phase was extracted with methylene chloride(2×20 mL) and chloroform (20 mL). The combined organic phases were driedover magnesium sulfate, filtered and evaporated under reduced pressureto afford 8-methoxy-1H-quinolin-2-one as white powder with quantitativeyield (1.9 g).

Step 2

To a solution of 8-methoxy-1H-quinolin-2-one prepared in step 1 (430 mg,2.4 mmol, 1.0 eq) in ethanol (40 mL) was added rhodium on aluminapowder. The suspension was hydrogenated under 4 bars of dihydrogen for 4hours at 30° C. Then, the suspension was filtered over celite andevaporated under vacuum to afford a 70/30 mixture of8-methoxy-3,4-dihydro-1H-quinolin-2-one and starting material. Themixture (430 mg) was used crude in the next step without purification.

Step 3

3-hex-5-ynyl-8-methoxy-3,4-dihydro-1H-quinolin-2-one was preparedaccording to General Procedure B using8-methoxy-3,4-dihydro-1H-quinolin-2-one prepared in step 2 (430 mg, 2.4mmol) and 6-iodo-1-hexyne (960 μL, 7.3 mmol). The expected compound wasisolated as light yellow powder (231 mg).

Step 4

The title compound was prepared according to General Procedure D, using3-hex-5-ynyl-8-methoxy-3,4-dihydro-1H-quinolin-2-one prepared in step 3(100 mg, 0.4 mmol) and Key Intermediate I (119 mg, 0.4 mmol). Theexpected compound was isolated as beige powder after flashchromatography and lyophilization with 69% yield (145 mg).

¹H NMR (DMSO): 8.97 (s, 1H), 7.36 (s, 1H), 7.05 (dd, J=8.5 and 11.3 Hz,1H), 6.92-6.74 (m, 5H), 5.27 (s, 1H), 4.39 (s, 2H), 3.75 (m, 5H), 2.92(dd, J=5.7 and 15.6 Hz, 1H), 2.63 (m, 1H), 2.32 (m, 3H), 1.98 (m, 1H),1.75 (m, 1H), 1.63 (m, 1H), 1.41 (m, 2H), 1.28 (m, 3H), 1.15 (m, 1H),0.66 (t, J=7.2 Hz, 3H), 0.53 (m, 2H), 0.28 (m, 2H)

Example 132-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-butyl)-4H-pyrido[3,2-b][1,4]oxazin-3-one

Step 1

To a stirred solution of trimethylsilylacetylene (595 μL, 4.2 mmol, 3.0eq) in dry tetrahydrofurane was added at −78° C. a 2 M solution ofn-butyllithium in hexane (2.4 mL, 4.9 mmol, 3.5 eq). After 2 minutes,hexamethylphosphoramide (0.64 mL) and2-(4-bromo-butyl)-4H-pyrido[3,2-b][1,4]oxazin-3-one (400 mg, 1.4 mmol,1.0 eq) were added. The mixture was stirred from −78° C. to roomtemperature during 18 hours. The mixture was then quenched with water(10 mL) and extracted with methylene chloride (3×10 mL). The combinedorganic phases were dried over magnesium sulfate, filtered andevaporated under reduced pressure. The LC/MS analysis of the orange oilobtained showed a mixture of sillylated compound and terminal alkyne.

The mixture was solubilized in THF and a 1 M solution oftetrabutylammonium fluoride in tetrahydrofurane (2.8 mL, 2.8 mmol, 2.0eq) was added. The mixture was stirred at room temperature for 18 hours.Water (10 mL) was added and the mixture was extracted with ethyl acetate(3×10 mL). The combined organic phases were dried over magnesiumsulfate, filtered and evaporated under reduced pressure. The residue waspurified by flash chromatography using cyclohexane and ethyl acetate(100/0 to 80/20) to afford2-hex-5-ynyl-4H-pyrido[3,2-b][1,4]oxazin-3-one with an overall yield of19% (60 mg).

Step 2

The expected compound was prepared according to General Procedure D,using 2-hex-5-ynyl-4H-pyrido[3,2-b][1,4]oxazin-3-one prepared in step 1(60 mg, 0.3 mmol) and Key Intermediate 1 (73 mg, 0.3 mmol). After theflash chromatography, the compound was purified by preparative HPLC toafford after lyophilization the expected compound as light blue solidwith 38% yield (51 mg).

¹H NMR (CDCl₁): 7.98 (d, 4.5 Hz, 1H), 7.35 (s, 1H), 7.32 (d, J=8.0 Hz,1H), 6.98 (m, 2H), 6.91 (dd, J=2.2 and 8.1 Hz, 1H), 6.76 (m, 1H), 4.62(m, 1H), 4.44 (dd, J=2.1 and 14.0 Hz, 1H), 4.33 (d, J=14.0 Hz, 1H), 3.78(d, J=7.0 Hz, 2H), 2.31 (m, 2H), 1.97 (m, 3H), 1.81 (m, 1H), 1.54 (m,4H), 1.22 (m, 2H), 0.82 (t, J=7.3 Hz, 3H), 0.63 (m, 2H), 0.32 (m, 2H)

Example 146-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one

Step 1

In a sealed tube, to a suspension of6-bromo-3,4-dihydro-1H-[1,8]naphthyridin-2-one (800 mg, 3.5 mmol, 1.0eq) in toluene (20 mL) were added successively sodium carbonate (746 mg,7.0 mmol, 2.0 eq), tributyl(vinyl)tin (1.3 g, 4.2 mmol, 1.2 eq) andwater (1 mL). The suspension was degazed with argon andtetrakis(triphenylphosphine)palladium(0) (407 mg, 0.3 mmol, 0.1 eq) wasadded. The reaction mixture was stirred at 120° C. during 12 hours.After cooling down, a saturated solution of sodium hydrogenocarbonate(10 mL) was added and the mixture was extracted with ethyl acetate (2×10mL) and methylene chloride (10 mL). The combined organic phases weredried over magnesium sulfate, filtered and evaporated under reducedpressure. The crude residue was purified by flash chromatography usingcyclohexane and ethyl acetate (100/0 to 50/50). The residue obtainedafter solvent evaporation was precipitated in methylene chloride andn-pentane to afford 6-vinyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one aswhite powder with 75% yield (460 mg).

Step 2

To a solution of 6-vinyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one preparedin step 1 (310 mg, 1.8 mmol, 1.0 eq) in methylene chloride (15 mL) wasadded 4-bromo-1-butene (360 μL, 3.6 mmol, 2.0 eq). The solution wasdegassed with argon before the addition of Grubbs' catalyst (secondgeneration) (75 mg, 0.09 mmol, 0.05 eq). The reaction mixture was heatedat 50° C. during 4 hours. After cooling down, a saturated solution ofsodium hydrogenocarbonate (10 mL) was added and the aqueous phase wasextracted with methylene chloride (15 mL) and ethyl acetate (2×15 mL).The combined organic phases were dried over magnesium sulfate, filteredand evaporated under reduced pressure. The crude residue was purified byflash chromatography using cyclohexane and ethyl acetate (100/0 to50/50) to afford6-((E)-4-bromo-but-1-enyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one aswhite powder with 66% yield (330 mg).

Step 3

To a solution of6-((E)-4-bromo-but-1-enyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-oneprepared in step 2 (330 mg, 1.2 mmol, 1.0 eq) in ethanol (40 mL) wasadded rhodium on alumina powder. The suspension was hydrogenated under 1bar of dihydrogen during 4 hours at 15° C. Then, the suspension wasfiltered over celite and evaporated under vacuum. The crude residue waspurified by flash chromatography using cyclohexane and ethyl acetate(100/0 to 50/50) to afford6-(4-bromo-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one as white powderwith 54% yield (220 mg).

Step 4

To a solution of trimethylsilylacetylene (1.1 μL, 7.8 mmol, 10.0 eq) intetrahydrofuran (10 mL) was added at −78° C. a 2 M solution ofn-butyllithium in cyclohexane (1.6 mL, 3.1 mmol, 4.0 eq) and HMPA (0.5mL, 3.1 mmol, 4.0 eq). After 5 minutes, a solution of6-(4-bromo-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one prepared instep 3 (220 mg, 0.8 mmol, 1.0 eq) in tetrahydrofuran (10 mL) was addedat −78° C. The reaction mixture was stirred from −78° C. to roomtemperature for 18 hours. A saturated solution of sodiumhydrogenocarbonate (15 mL) was added and the aqueous phase was extractedwith ethyl acetate (3×15 mL). The combined organic phases were driedover magnesium sulfate, filtered and evaporated under reduced pressure.The crude residue was purified by flash chromatography using cyclohexaneand ethyl acetate (100/0 to 50/50) to afford6-(6-trimethylsilanyl-hex-5-ynyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-oneas white powder (210 mg) in mixture with traces of HMPA. This mixturewas used in the next step.

Step 5

To a solution of6-(6-trimethylsilanyl-hex-5-ynyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one(210 mg, 0.7 mmol, 1.0 eq) in tetrahydrofuran (10 mL) was added a 1Msolution of tetra-n-butylammonium fluoride in tetrahydrofuran (2.1 mL,2.1 mmol, 3.0 eq). After 12 hours at room temperature, water (10 mL) wasadded and the reaction mixture was extracted with ethyl acetate (3×10mL). The combined organic phases were dried over magnesium sulfate,filtered and evaporated under reduced pressure. The crude residue waspurified by flash chromatography using cyclohexane and ethyl acetate(100/0 to 40/60) to afford6-hex-5-ynyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one as white powder (100mg) with an overall yield of 56% on step 4 and step 5.

Step 6

The title compound was prepared according to General Procedure D, using6-hex-5-ynyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one prepared in step 5(100 mg, 0.4 mmol) and Key Intermediate I (122 mg, 0.4 mmol). Theexpected compound was isolated as white powder after flashchromatography and lyophilization with 56% yield (125 mg).

¹H NMR (DMSO): 10.30 (s, 1H), 7.89 (d, J=2.0 Hz, 1H), 7.39 (s, 1H), 7.35(s, 1H), 7.06 (dd, J=8.5 and 11.4 Hz, 1H), 6.92 (dd, J=2.0 and 8.5 Hz,1H), 6.79 (m, 1H), 5.28 (s, 1H), 4.39 (s, 2H), 3.75 (d, J=7.0 Hz, 2H),2.82 (t, J=7.5 Hz, 2H), 2.45 (m, 4H), 2.32 (m, 2H), 1.98 (m, 1H), 1.76(m, 1H), 1.44 (m, 4H), 1.14 (m, 1H), 0.67 (t, J=7.2 Hz, 3H), 0.52 (m,2H), 0.26 (m, 2H).

Example 156-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one

Step 1

In a sealed tube, to a suspension of6-bromo-3,4-dihydro-1H-[1,8]naphthyridin-2-one (800 mg, 3.5 mmol, 1.0eq) in toluene (20 mL) were added successively sodium carbonate (746 mg,7.0 mmol, 2.0 eq), tributyl(vinyl)tin (1.3 g, 4.2 mmol, 1.2 eq) andwater (1 mL). The suspension was degazed with argon andtetrakis(triphenylphosphine)palladium(0) (407 mg, 0.3 mmol, 0.1 eq) wasadded. The reaction mixture was stirred at 120° C. during 12 hours.After cooling down, a saturated solution of sodium hydrogenocarbonate(10 mL) was added and the mixture was extracted with ethyl acetate (2×10mL) and methylene chloride (10 mL). The combined organic phases weredried over magnesium sulfate, filtered and evaporated under reducedpressure. The crude residue was purified by flash chromatography usingcyclohexane and ethyl acetate (100/0 to 50/50). The residue obtainedafter solvent evaporation was precipitated in methylene chloride andn-pentane to afford 6-vinyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one aswhite powder with 75% yield (460 mg).

Step 2

To a solution of 6-vinyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one preparedin step 1 (310 mg, 1.8 mmol, 1.0 eq) in methylene chloride (15 mL) wasadded 4-bromo-1-butene (360 μL, 3.6 mmol, 2.0 eq). The solution wasdegased with argon before the addition of Grubbs' catalyst (secondgeneration) (75 mg, 0.09 mmol, 0.05 eq). The reaction mixture was heatedat 50° C. during 4 hours. After cooling down, a saturated solution ofsodium hydrogenocarbonate (10 mL) was added and the aqueous phase wasextracted with methylene chloride (15 mL) and ethyl acetate (2×15 mL).The combined organic phases were dried over magnesium sulfate, filteredand evaporated under reduced pressure. The crude residue was purified byflash chromatography using cyclohexane and ethyl acetate (100/0 to50/50) to afford6-((E)-4-bromo-but-1-enyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one aswhite powder with 66% yield (330 mg).

Step 3

To a solution of6-((E)-4-bromo-but-1-enyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-oneprepared in step 2 (330 mg, 1.2 mmol, 1.0 eq) in ethanol (40 mL) wasadded rhodium on alumina powder. The suspension was hydrogenated under 1bar of dihydrogen during 4 hours at 15° C. Then, the suspension wasfiltered over celite and evaporated under vacuum. The crude residue waspurified by flash chromatography using cyclohexane and ethyl acetate(100/0 to 50/50) to afford6-(4-bromo-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one as white powderwith 54% yield (220 mg).

Step 4

To a solution of trimethylsilylacetylene (1.1 μL, 7.8 mmol, 10.0 eq) intetrahydrofuran (10 mL) was added at −78° C. a 2 M solution ofn-butyllithium in cyclohexane (1.6 mL, 3.1 mmol, 4.0 eq) and HMPA (0.5mL, 3.1 mmol, 4.0 eq). After 5 minutes, a solution of6-(4-bromo-butyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one prepared instep 3 (220 mg, 0.8 mmol, 1.0 eq) in tetrahydrofuran (10 mL) was addedat −78° C. The reaction mixture was stirred from −78° C. to roomtemperature for 18 hours. A saturated solution of sodiumhydrogenocarbonate (15 mL) was added and the aqueous phase was extractedwith ethyl acetate (3×15 mL). The combined organic phases were driedover magnesium sulfate, filtered and evaporated under reduced pressure.The crude residue was purified by flash chromatography using cyclohexaneand ethyl acetate (100/0 to 50/50) to afford6-(6-trimethylsilanyl-hex-5-ynyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-oneas white powder (210 mg) in mixture with traces of HMPA. This mixturewas used in the next step.

Step 5

To a solution of6-(6-trimethylsilanyl-hex-5-ynyl)-3,4-dihydro-1H-[1,8]naphthyridin-2-one(210 mg, 0.7 mmol, 1.0 eq) in tetrahydrofuran (10 mL) was added a 1Msolution of tetra-n-butylammonium fluoride in tetrahydrofuran (2.1 mL,2.1 mmol, 3.0 eq). After 12 hours at room temperature, water (10 mL) wasadded and the reaction mixture was extracted with ethyl acetate (3×10mL). The combined organic phases were dried over magnesium sulfate,filtered and evaporated under reduced pressure. The crude residue waspurified by flash chromatography using cyclohexane and ethyl acetate(100/0 to 40/60) to afford6-hex-5-ynyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one as white powder (100mg) with an overall yield of 56% on step 4 and step 5.

Step 6

The title compound was prepared according to General Procedure D, using6-hex-5-ynyl-3,4-dihydro-1H-[1,8]naphthyridin-2-one prepared in step 5(100 mg, 0.4 mmol) and Key Intermediate I (122 mg, 0.4 mmol). Theexpected compound was isolated as white powder after flashchromatography and lyophilization with 56% yield (125 mg).

¹H NMR (DMSO): 10.30 (s, 1H), 7.89 (d, J=2.0 Hz, 1H), 7.39 (s, 1H), 7.35(s, 1H), 7.06 (dd, J=8.5 and 11.4 Hz, 1H), 6.92 (dd, J=2.0 and 8.5 Hz,1H), 6.79 (m, 1H), 5.28 (s, 1H), 4.39 (s, 2H), 3.75 (d, J=7.0 Hz, 2H),2.82 (t, J=7.5 Hz, 2H), 2.45 (m, 4H), 2.32 (m, 2H), 1.98 (m, 1H), 1.76(m, 1H), 1.44 (m, 4H), 1.14 (m, 1H), 0.67 (t, J=7.2 Hz, 3H), 0.52 (m,2H), 0.26 (m, 2H)

Example 163-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-butyl)-8-methoxy-3,4-dihydro-1H-quinolin-2-one

Step 1

To a solution of 2-chloro-8-methoxy-quinoline (2.1 g, 10.7 mmol, 1.0 eq)in acetic acid (15 mL) was added water (5 mL). The mixture was stirredat 100° C. during 18 hours. After cooling down, the solvent wasevaporated. Water (30 mL) and a 25% solution of ammonium hydroxide (20mL) were added. The aqueous phase was extracted with methylene chloride(2×20 mL) and chloroform (20 mL). The combined organic phases were driedover magnesium sulfate, filtered and evaporated under reduced pressureto afford 8-methoxy-1H-quinolin-2-one as white powder with quantitativeyield (1.9 g).

Step 2

To a solution of 8-methoxy-1H-quinolin-2-one prepared in step 1 (430 mg,2.4 mmol, 1.0 eq) in ethanol (40 mL) was added rhodium on aluminapowder. The suspension was hydrogenated under 4 bars of dihydrogen for 4hours at 30° C. Then, the suspension was filtered over celite andevaporated under vacuum to afford a 70/30 mixture of8-methoxy-3,4-dihydro-1H-quinolin-2-one and starting material. Themixture (430 mg) was used crude in the next step without purification.

Step 3

3-hex-5-ynyl-8-methoxy-3,4-dihydro-1H-quinolin-2-one was preparedaccording to General Procedure B using8-methoxy-3,4-dihydro-1H-quinolin-2-one prepared in step 2 (430 mg, 2.4mmol) and 6-iodo-1-hexyne (960 μL, 7.3 mmol). The expected compound wasisolated as light yellow powder (231 mg).

Step 4

The title compound was prepared according to General Procedure D, using3-hex-5-ynyl-8-methoxy-3,4-dihydro-1H-quinolin-2-one prepared in step 3(100 mg, 0.4 mmol) and Key Intermediate I (119 mg, 0.4 mmol). Theexpected compound was isolated as beige powder after flashchromatography and lyophilization with 69% yield (145 mg).

¹H NMR (DMSO): 8.97 (s, 1H), 7.36 (s, 1H), 7.05 (dd, J=8.5 and 11.3 Hz,1H), 6.92-6.74 (m, 5H), 5.27 (s, 1H), 4.39 (s, 2H), 3.75 (m, 5H), 2.92(dd, J=5.7 and 15.6 Hz, 1H), 2.63 (m, 1H), 2.32 (m, 3H), 1.98 (m, 1H),1.75 (m, 1H), 1.63 (m, 1H), 1.41 (m, 2H), 1.28 (m, 3H), 1.15 (m, 1H),0.66 (t, J=7.2 Hz, 3H), 0.53 (m, 2H), 0.28 (m, 2H)

Example 173-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-butyl)-1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one

Step 1

The 3-hex-5-ynyl-1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one was preparedaccording to General Procedure B using1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one (350 mg, 2.6 mmol, 1.0 eq) and6-iodo-1-hexyne (310 μL, 2.3 mmol, 0.9 eq). The expected compound wasisolated as white powder with 18% yield (100 mg).

Step 2

The expected compound was prepared according to General Procedure D,using 3-hex-5-ynyl-1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one prepared instep 1 (100 mg, 0.5 mmol) and Key Intermediate I (130 mg, 0.5 mmol). Theexpected compound was isolated as white powder after flashchromatography and lyophilization with 33% yield (77 mg).

¹H NMR (DMSO): 10.93 (s, 1H), 8.03 (d, J=4.5 Hz, 1H), 7.55 (d, J=7.0 Hz,1H), 7.32 (s, 1H), 7.04 (dd, J=9.0 and 11.5 Hz, 1H), 6.93 (m, 2H), 6.82(m, 1H), 5.25 (s, 1H), 4.38 (s, 2H), 3.75 (d, J=7.0 Hz, 2H), 3.50 (t,J=5.8 Hz, 1H), 2.30 (m, 2H), 2.00-1.65 (m, 4H), 1.40 (m, 2H), 1.16 (m,3H), 0.65 (t, J=7.2 Hz, 3H), 0.53 (m, 2H), 0.28 (m, 2H).

Example 18 Biological Methods A. Drugs, Reagents and Cell Lines

PCI 10213, 10214 and 10216 are suspended in DMSO at a concentration of100 mmol/L, fluorodeoxyuridine (FUdR) that can be obtained from Sigma(St Louis, Mo.) and maintained in sterile double-distilled water atstock concentrations of 50 mmol/L. PCI 10216 has the structure:

Recombinant human deoxyuridine nucleotidohydrolase (dUTPase) isexpressed and purified as described in Ladner R D, Carr S A, HuddlestonM J, McNulty D E, Caradonna S J. J Biol Chem. 1996 Mar. 29; 271(13):7752-7. All drugs stocks were aliquoted and diluted as appropriateprior to use. The oligonucelotide primer, templates and fluorophore- andquencher-labeled detection probes are synthesized by Integrated DNATechnologies (Coralville, Iowa), subjected to polyacrylamide gelelectrophoresis purification and reconstituted in Omnipur sterilenuclease-free water (EMD Chemicals USA, Gibbstown N.J.) at a stockconcentration of 100 μmol/L. The two non-emissive (dark) quenchingmolecules incorporated into the detection probes include the Iowa blackfluorescein quencher (IBFQ; absorption max 531 nm) and ZEN(non-abbreviation; absorption max 532 nm). The fluorescent labelutilized was 6-FAM (5′-carboxyfluorescein; excitation max.=494 nm,emission max.=520 nm). Probes were further diluted to a working stock of10 μmol/L and aliquoted to avoid repeated freeze/thaw cycles. AmpliTaqGold DNA Polymerase, GeneAmp 10×PCR Buffer 2, MgCl₂ and MicroAmp Optical96-well Reaction Plates were purchased from Applied Biosystems(Carlsbad, Calif.). dNTPs were purchased individually at stockconcentrations of 100 mmol/L from New England Biolabs atHPLC-certified >99% purity (Ipswich, Mass.).

B. Assay Components, Instrumentation and Real-Time FluorescenceConditions

Reaction mixtures contained primer, probe and template at an equimolarfinal concentration of 0.4 μmol/L. Magnesium chloride (MgCl₂) wasincluded at a final concentration of 2 mmol/L. Non-limiting dNTPs wereincluded in the reaction mix in excess at a final concentration of 100μmol/L (dUTP/dTTP was excluded). AmpliTaq Gold DNA polymerase was addedat 0.875 U/reaction, 2.5 μl of 10×PCR buffer 2 added and nuclease-freeddH₂O added to a final reaction volume of 25 μl. For dUTP inhibitionanalysis, the volume of ddH₂O was further modified to accommodate anadditional 1 μl of dUTPase (10 ng/μl) and 1 μl of inhibitor or DMSOcontrol. Thermal profiling and fluorescence detection was performedusing the ‘isothermal’ program on board an Applied Biosystems 7500Real-Time PCR System. For analysis of dNTPs, the thermal profileconsisted of an 8 min 37° C. step followed by a 10 min 95° C. step to‘hot-start’ the Taq polymerase and a primer extension time of up to 30min at 60° C. depending on the application. Raw fluorescence spectra for6-FAM was measured using filter A at specified time intervals to followassay progression using Sequence Detection Software (SDS Version 1.4,Applied Biosystems) and exported and analyzed in Microsoft Excel(Microsoft, Redmond Wash.) and Prism (GraphPad Software, La JollaCalif.). In all cases, fluorescence values for blank reactions (limitingdNTP omitted) were subtracted to give normalized fluorescence units(NFU) to account for background fluorescence.

C. MTS Growth Inhibition Assay

The Cell Titer⁹⁶ AQueous MTS assay (Promega) was carried out accordingto the manufacturers guidelines. IC_(50(72h)) values were calculatedfrom sigmoidal-dose response curves utilizing Prism (Graphpad, SanDiego, Calif.). The combination effect was determined by the combinationindex (CI) method utilizing Calcusyn software (Biosoft, Ferguson, Mo.).Fraction affected (FA) was calculated from the percent growthinhibition: FA=(100−% growth inhibition)/100. CI values <1, synergism;1-1.2, additive and >1.2, antagonism.

D. Colony Formation Assay

Colony forming assay showing the ability of colon (SW620, HCT116),non-small cell lung (A549, H460, H1299 and H358) and breast (MCF7)cancer cells to survive and proliferate following transient 24 hourexposure to single agent PCI 1013, FUdR and combinations. Specifically,cells were seeded at densities between 50 and 100 cells/well in 24-wellplates. Twenty-four hours later, cells were treated with increasingconcentrations of PCI 10213, a fixed dose of FUdR and combinations ofthese. After 24 hours, drug was removed, cells were rinsed and allowedto outgrow for 10-14 days. At the conclusion of the outgrowth, cellswere fixed in 60% ice cold methanol and stained with 0.1% crystalviolet, scanned and counted. Data is presented as percentage ofuntreated controls (mean±SD). Fraction affected and combination indexeswere calculated according to the method of Chou and Talalay where <1 isindicative of a synergistic drug interaction.

E. In Vivo Analysis

Xenograft experiments were conducted in male NU/NU nude mice (CharlesRiver, Wilmington, Mass.) that were 6-8 weeks old. Subcutaneous A549xenografts were established and allowed to grow until they reached ˜50mm³ (day 1). Animals were randomized to treatment groups: vehicle,pemetrexed 50 mg/kg, PCI 10213 and combination of pemetrexed plus PCI10213 (n=5, group). Pemetrexed was administered at 50 mg/kg byintraperitoneal injection every two days. PCI 10213 was administered at75 mg/kg by intraperitoneal injection every two days. The combination ofpemetrexed and PCI 10214 was administered by intraperitoneal injectionevery two days. Two perpendicular diameters of tumors were measuredevery 2 days with a digital caliper by the same investigator. Tumorvolume was calculated according to the following formula: TV(mm³)=(length[mm]×(width[mm]²)/2. Mice were inspected everyday foroverall health and bodyweight was measured every 2 days as an index oftoxicity. All animal protocols were approved by the USC InstitutionalAnimal Care and Use Committee (IACUC).

Example 19 Identification of the dUTPase Inhibitor PCI 10213

TABLE 1 % Inhibition umol/L 10216 10213 83.3 95.4 86.1 41.7 97.2 73.820.8 101.1 64.5 10.4 95.9 60.3 5.2 84.6 40.9 2.6 76.6 31.6 1.3 60.4 28.0

PCI 10213 and reference compound 10216 were screened in afluorescence-based assay. The assay employs a DNA polymerase-basedapproach utilizing an oligonucleotide template with 3 distinct regions:a 3′ primer binding region, a mid-template dUTP/thymidine triphosphate(TTP) detection region and a 5′ 6-Flavin adenine mononucleotide(FAM)-reaction, the probe and primer hybridize to the oligonucleotidetemplate to form the template:primer:probe complex. When Taq polymerasebinds to the primer in the TPP complex and dUTP is present, successfulextension of the nascent strand occurs and the inherent 5′ to 3′exonuclease activity of Taq polymerase cleaves and displaces the6-FAM-labeled probe in a 5′ to 3′ direction, releasing the 6-FAMfluorophore from its proximity to the three quenchers. This displacementeffectively disrupts the Förster resonance energy transfer (FRET) andthe resulting fluorescence detected upon excitation is directlyproportional to the amount of the dUTP available in the assay forincorporation (FIG. 3). Conversely, when the dUTP is unavailable,exhausted, or degraded by dUTPase and is no longer available forincorporation, Taq polymerase stalls and extension delay and/or chaintermination of the nascent strand occurs. In this instance, probehydrolysis/degradation does not occur and the probe remains dark asfluorescence remains quenched via FRET. Since fluorescence is directlyproportional to the concentration of dUTP, the assay was easily modifiedto measure dUTP and the effects of inhibitors on dUTP hydrolysis by theenzyme dUTPase. The template BHQ-DT6 (Black Hole Quencher—DetectionTemplate 6) for detecting up to 60 pmols of dUTP was included for thisapplication of the assay along with 50 pmols of dUTP and 5 ng ofrecombinant dUTPase. The reaction was incubated at 37° C. for 8 mins andterminated by a 10 min incubation at 95° C. to simultaneously inactivatedUTPase and activate the hot-start Taq polymerase. The fluorescencegenerated during the detection step is directly proportional to theconcentration of dUTP remaining after the 8 min incubation. Theconcentration of dUTP at reaction termination and therefore inhibitionof dUTPase in the presence and absence of inhibitors and appropriatedimethyl sulfoxide (DMSO) controls can be determined. In preliminarydUTPase inhibition experiments PCI 10213 was compared directly to PCI10216 at a range of concentrations between 0 and 83 μmol/L (Table 1,FIG. 3A). Inhibition of dUTPase enzymatic activity at the maximum doseof 83 μmol/L was significant for both compounds at 95 and 86% for PCI10216 and 10213 respectively. The level of inhibition at 1.3 μmol/L was60% and 28% respectively. The IC₅₀ calculated in Prism for PCI 10216 was0.8 μmol/L and for PCI 10213 7.2 μmol/L.

Example 20 PCI 10213 Shows Little to No Single Agent Activity inContrast to PCI 10216

PCI 10213, 10214 and 10216 were evaluated for their antitumor activityin colorectal cancer cells using the MTS growth inhibition assay.

HCT116 and SW620 cells were exposed to increasing concentrations of eachagent for 72 hours and growth inhibition was directly compared tovehicle-treated controls. In HCT116 cells, PCI 10213 and PCI 10214demonstrated little to no single agent activity even up to the elevatedconcentration of 75 μmol/L with a modest decrease in growth of 28%observed with 100 μmol/L PCI 10213. In contrast, PCI 10216 demonstrateddose-dependent decreases in cell proliferation detectable atconcentrations as low as 6.25 μmol/L and culminating with a 67%reduction in proliferation with 100 μmol/L.

In SW620 cells, neither PCI 10213 or 10214 had any single agent activityup to 75 μmol/L and only modest activity of ˜30% at 100 μmol/L. PCI10216 demonstrated dose-dependent decreases in proliferation up to 70%at 100 μmol/L (FIG. 3B).

The NSCLC cell lines A549 and H1299 were exposed to increasingconcentrations of each agent for 72 hours and growth inhibition wasdirectly compared to vehicle-treated controls. In A549 cells, PCI 10213and PCI 10214 demonstrated modest single agent activity with theelevated concentration of 75 and 100 μmol/L showing modest decreases ingrowth of ˜25% at 100 μmol/L PCI 10213 and 10214. PCI 10216 demonstratedsimilar decreases in cell proliferation as 10213 and 10214 at lowerdoses, but significantly more at the elevated doses with 30% and 55%reductions in proliferation with 75 and 100 mol/L respectively. In H1299cells, PCI 10213, 10214 and 10216 had modest single agent activity up to12.5 μmol/L with increased activity of up to ˜40% at 100 μmol/L. Ofnote, PCI 10213 demonstrated greater growth inhibition at 100 μmol/Lthan PCI 10216 with decreases in cell proliferation of 40% and 30%respectively at 100 μmol/L.

Example 21 PCI 10213 Demonstrates Synergy with 5-FU Through IncreaseGrowth Inhibition

MTS growth inhibition assays were performed to evaluate theeffectiveness of both PCI 10213 and reference compound PCI 10216 aloneand in combination with the fluoropyrimidine thymidylate synthase (TS)inhibitor 5-fluorouracil (5-FU) at inhibiting the growth of colorectal(HCT116 and SW620) cell line models. Increasing concentrations of 5-FUbetween 0 and 100 μmol/L demonstrated dose-dependent increases in growthinhibition in both the colorectal cancer cell lines evaluated.Simultaneous treatment with increasing concentrations of 5-FU and eitherPCI 10213 and 10216 at fixed concentrations of 25 μmol/L resulted inadditive and synergistic increases in growth inhibition over themajority of concentrations tested up to 25 μmol/L 5-FU in both CRC celllines examined. Of note, PCI 10216 as a single agent used at 25 μmol/Linduced 30% growth inhibition in SW620 cells and 44% in HCT116 cellswhereas PCI 10213 had no detectable effect on growth inhibition at 25μmol/L in either cell line despite showing additive and synergisticinteractions with 5-FU. These data demonstrate a clear enhancement of5-FU growth inhibitory activity through the addition of PCI 10213 withsignificantly less single agent activity than PCI 10216. See, FIG. 4.

Example 22 PCI 10213 Demonstrates Synergy with FUdR in Reducing CancerCell Viability

Colony forming assays were performed to evaluate the effectiveness ofboth PCI 10213, PCI 10214 and reference compound PCI 10216 alone and incombination with the fluoropyrimidine thymidylate synthase (TS)inhibitor fluorodeoxyuridine (FUdR) at reducing cancer cell viability incolorectal (HCT116), breast (MCF-7) and non-small cell lung (H1299,A549, H358 and H460) cell line models. Increasing concentrations of FUdRbetween 0.5 and 2.5 μmol/L demonstrated dose-dependent decreases incolonies formed in all cell lines evaluated. Increasing concentrationsof PCI 10213 between 3.1 and 25 μmol/L had no significant effects on thenumber of colonies formed whereas the elevated concentration of PCI10216 at 25 and 50 μmol/L demonstrated some reduction in the number ofcolonies formed in A549, H460 and HCT116 cells. Reference compound PCI10216 demonstrated strong synergy when combined with fixed doses of FUdRin all cell lines examined. Subsequently, increasing concentrations ofPCI 10213 were combined with a fixed dose of FUdR to evaluate thecombined drug effect. In NSCLC cells, concentrations of PCI 10213ranging from 3.1 μmol/L to 25 μmol/L were combined with 1 μmol/L FUdR.One jμmol/L FUdR had no significant effect on number of colonies formedcompared to vehicle-treated controls. However, all combinations of PCI10213 and 1 μmol/L FUdR demonstrated highly significant reductions incolonies formed when compared to the corresponding single agentconcentrations of PCI 10213 alone or 1 μmol/L FUdR. The effectiveness ofthis combination was pronounced in A549 and H460 cells where 12.5 and 25μmol/L PCI 10213 combined with 1 μmol/L FUdR reduced cell viabilityby >95% compared to vehicle-treated controls.

In colorectal cancer cells, concentrations of PCI 10213 ranging from 3.1μmol/L to 50 μmol/L were combined with 0.5 μmol/L FUdR in HCT116 cellsand 1 μmol/L FUdR in SW620 cells.

Similar to the NSCLC cells, neither PCI 10213 or the fixed dose of FUdRsingle agent had any significant effect on the number of colonies formedbut demonstrated a strong synergistic reduction in colonies in allcombinations tested in HCT116 and SW620 cells. Specifically, thecombination of 12.5 and 25 μmol/L PCI 10213 combined with 0.5 μmol/LFUdR reduced colony formation by >95% in HCT116 cells and >50% in thestrongly FUdR-resistant SW620 cell line. Importantly, despite neitheragent alone exerting any effect on the number of colonies formed,complete loss of viability was achieved with the 50 μmol/L PCI 10213 andFUdR combination in HCT116 cells. In the MCF-7 breast cancer cell line,treatment with 0.5 μmol/L FUdR had no significant impact on the numberof colonies formed, but when combined with concentrations of PCI 10213ranging from 3.1 μmol/L to 25 μmol/L significant reductions in colonyformation of between 30 and 60% was observed. See, FIGS. 5-7.

Example 23 Diastereomer PCI 10586 is the Primary Active Component of PCI10213 A. Drugs and Reagents

PCI 10586, 10585 and 10213 were suspended in DMSO at a concentration of100 mmol/L, fluorodeoxyuridine (FUdR) was obtained from Sigma (St Louis,Mo.) and maintained in sterile double-distilled water at stockconcentrations of 50 mmol/L. Recombinant human deoxyuridinenucleotidohydrolase (dUTPase) was expressed and purified as describedpreviously [Please provide reference or confirm as noted above]. Alldrugs stocks were aliquoted and diluted as appropriate prior to use. Theoligonucelotide primer, templates and fluorophore- and quencher-labeleddetection probes were synthesized by Integrated DNA Technologies(Coralville, Iowa), subjected to polyacrylamide gel electrophoresispurification and reconstituted in Omnipur sterile nuclease-free water(EMD Chemicals USA, Gibbstown N.J.) at a stock concentration of 100μmol/L. The two non-emissive (dark) quenching molecules incorporatedinto the detection probes include the Iowa black fluorescein quencher(IBFQ; absorption max 531 nm) and ZEN (non-abbreviation; absorption max532 nm). The fluorescent label utilized was 6-FAM(5′-carboxyfluorescein; excitation max.=494 nm, emission max.=520 nm).Probes were further diluted to a working stock of 10 μmol/L andaliquoted to avoid repeated freeze/thaw cycles. AmpliTaq Gold DNAPolymerase, GeneAmp 10×PCR Buffer 2, MgCl₂ and MicroAmp Optical 96-wellReaction Plates were purchased from Applied Biosystems (Carlsbad,Calif.). dNTPs were purchased individually at stock concentrations of100 mmol/L from New England Biolabs at HPLC-certified >99% purity(Ipswich, Mass.).

B Assay Components, Instrumentation and Real-Time FluorescenceConditions

Reaction mixtures contained primer, probe and template at an equimolarfinal concentration of 0.4 μmol/L. MgCl₂ was included at a finalconcentration of 3 mmol/L. Non-limiting dNTPs were included in thereaction mix in excess at a final concentration of 100 μmol/L (dUTP/dTTPwas excluded). AmpliTaq Gold DNA polymerase was added at 0.875U/reaction, 2.5 μl of 10×PCR buffer 2 added and nuclease-free ddH₂Oadded to a final reaction volume of 30 μl. For dUTP inhibition analysis,the volume of ddH₂O was further modified to accommodate an additional 1μl of dUTPase (2.5 ng/μl) and 1 μl of inhibitor or DMSO control. Thermalprofiling and fluorescence detection was performed using the‘isothermal’ program on board an Applied Biosystems 7500 Real-Time PCRSystem. For analysis of dNTPs, the thermal profile consisted of an 10min 37° C. step followed by a 10 min 95° C. step to ‘hot-start’ the Taqpolymerase and a 5-cycle primer extension time of 10 min at 60° C. Rawfluorescence spectra for 6-FAM was measured using filter A at specifiedtime intervals to follow assay progression using Sequence DetectionSoftware (SDS Version 1.4, Applied Biosystems) and exported and analyzedin Microsoft Excel (Microsoft, Redmond Wash.) and Prism (GraphPadSoftware, La Jolla Calif.). In all cases, fluorescence values for blankreactions (limiting dNTP omitted) were subtracted to give normalizedfluorescence units (NFU) to account for background fluorescence.

C. dUTPase Inhibition Screening Reveals that Diastereomer PCI 10586 isthe Primary Active Component of PCI 10213

PCI 10213 possesses two molecular diastereomers: PCI 10586 and 10585.The diastereomer compounds were isolated by preparative chiral HPLC andscreened in a novel fluorescence-based assay as described in Wilson etal. (2011) Nucleic Acids Res., Sept. 1 39 (17). The assay employs a DNApolymerase-based approach utilizing an oligonucleotide template with 3distinct regions: a 3′ primer binding region, a mid-template dUTP/TTPdetection region and a 5′ 6-FAM-labeled probe binding region thatincorporates a black hole quenching moiety as previously described.Since fluorescence is directly proportional to the concentration ofdUTP, the assay was easily modified to measure dUTP and the effects ofinhibitors on dUTP hydrolysis by the enzyme dUTPase. The templateBHQ-DT6 for detecting up to 60 pmols of dUTP was included for thisapplication of the assay along with 50 pmols of dUTP and 2.5 ng ofrecombinant dUTPase. The reaction was incubated at 37° C. for 10 minsand terminated by a 10 min incubation at 95° C. to simultaneouslyinactivate dUTPase and activate the hot-start Taq polymerase. Thesubsequent fluorescence detection step involved five 10 min cycles at60° C. to completion. The fluorescence generated during the detectionstep is directly proportional to the concentration of dUTP remainingafter the 10 min incubation. The concentration of dUTP at reactiontermination is directly proportional to the extent of inhibition ofdUTPase in the presence and absence of inhibitors and appropriate DMSOcontrols.

TABLE 2 PCI 10586, PCI 10585, and PCI 10213 were screened at thespecified compound concentrations using a fluorescence-based dUTPaseinhibition assay to determine dUTPase enzyme inhibition % dUTPaseInhibition μmol/L PCI 10586 PCI 10585 PCI 10213 83.3 75.6 9.5 54.6 41.764.3 5.4 45.5 20.8 66.1 5.5 30.0 10.4 55.8 6.5 24.9 5.2 38.3 3.4 14.52.6 27.8 0.5 9.6 1.3 16.4 0.6 4.7

dUTPase inhibition comparisons were made between compounds PCI 10213,10585, and 10586 at a range of concentrations between 1.3 and 83.3itmol/L (Table 2). Inhibition of dUTPase enzymatic activity at themaximum dose of 83.3 μmol/L was significant for compound 10586 with75.6% inhibition, moderate for compound 10213 with 54.6% inhibition andmodest for compound 10585 with 9.5%. At moderate concentration of 5.2μmol/L, compound 10586 demonstrated strong inhibition of 38%, compound10213 had 14.5% and 10585 had 3.4% inhibition. The level of inhibitionat 1.3 μmol/L was 16.4%, 4.7%, and 0.6% for 10585, 10213 and 10585respectively. The strong dUTPase inhibition observed for 10586, theintermediate inhibition of the heterogeneous 10213 and the distinct lackof dUTPase inhibition by 10585 confirms that diasteromer PCI 10586(28.46 min retention time) is the primary active molecule in compoundPCI 10213.

D. PCI 10586 Demonstrated Synergy with FUdR

Colony forming assays were performed to evaluate the effectiveness ofboth PCI 10213, PCI 10585 and PCI 10586 alone and in combination withthe fluoropyrimidine thymidylate synthase (TS) inhibitorfluorodeoxyuridine (FUdR) at reducing cancer cell viability in HCT116colorectal cancer cells. Increasing concentrations of FUdR between 0.5and 5 μmol/L demonstrated dose-dependent decreases in colonies formed(FIG. 11A). Increasing concentrations of PCI 10213, 10585 or 10586between 0.78 and 12.5 μmol/L had no significant effects on the number ofcolonies formed (FIG. 11B).

To evaluate the combined drug effect, increasing concentrations of PCI10213, 01585 and 10586 were combined with a fixed dose of 0.5 μmol/LFUdR. Of note, 0.5 μmol/L FUdR had no significant effect on number ofcolonies formed compared to vehicle-treated controls (FIG. 11A). PCI10213, demonstrated significant reductions in colonies formed whencombined with FUdR at concentrations of 6.25 μmol/L and greater.However, all concentrations of PCI 10286 combined with 0.5 μmol/L FUdRdemonstrated reductions in colonies formed even at the lowest dose of0.78 μmol/L when compared to the corresponding single agentconcentrations of PCI 10586 and 0.5 μmol/L FUdR. Importantly, PCI 10585demonstrated no reductions in colony formation when combined with FUdRat any concentration up to 6.25 μmol/L with modest reductions at 12.5μmol/L (FIG. 11C). These data support the in vitro dUTPase inhibitorscreen demonstrating that PCI 10586 has significantly more cell-basedactivity than PCI 10213 and that PCI 10585 is significantly less potentthan either 10213 or 10586.

Reference compound PCI 10950:

demonstrated strong synergy when combined with fixed doses of FUdR inall cell lines examined. Subsequently, increasing concentrations of PCI102951 and PCI 10952 were combined with a fixed dose of 0.5 μmol/L FUdRto evaluate the combined drug effect. Substantial reductions in coloniesformed were observed when PCI 10951 was combined with 0.5 μmol/L FUdRcompared to the corresponding single agent concentrations of PCI aloneor 0.5 μmol/L FUdR. PCI 10952 demonstrated modest reductions in coloniesformed when combined with 0.5 μmol/L FUdR in HCT116 cells under theconditions tested.

Colony forming assays were subsequently performed to evaluate theeffectiveness of additional PCI compounds alone and in combination withFUdR at reducing cancer cell viability in the HCT-8 colorectal cell linemodel. FUdR at 1 μmol/L demonstrated no substantial effect on coloniesformed. Increasing concentrations of all PCI compounds between 1.56 and6.25 μmol/L also had no significant effects on the number of coloniesformed. Reference compound PCI 10950 demonstrated strong synergy whencombined with the fixed dose of FUdR. Subsequently, increasingconcentrations of PCI compounds were combined with a fixed dose of 1μmol/L FUdR to evaluate the combined drug effect. Substantial reductionsin colonies formed were observed when PCI 10951, PCI 10927, PCI 10928,PCI 10929, PCI 10930, and PCI 10933 was combined with 1 μmol/L FUdRcompared to the corresponding single agent concentrations in HCT-8cells.

Other compounds were also assayed employing the various assays describedherein, and can be assayed following these and other assays known to theskilled artisan.

It should be understood that although the present invention has beenspecifically disclosed by certain aspects, embodiments, and optionalfeatures, modification, improvement and variation of such aspects,embodiments, and optional features can be resorted to by those skilledin the art, and that such modifications, improvements and variations areconsidered to be within the scope of this disclosure.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. In addition, where featuresor aspects of the invention are described in terms of Markush groups,those skilled in the art will recognize that the invention is alsothereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. A compound of formula (I) or (II):

or a tautomer thereof, including any stereoisomer, enantiomer ordiastereoisomer, or a pharmaceutically acceptable salt of each thereof,wherein

is a uracil isostere; W is a bond or optionally substituted —CH₂—; W¹ isa bond, N, or an optionally substituted CH group; X is a bond, O, S,NR¹⁹, optionally substituted C₁-C₆ alkylene, optionally substitutedC₂-C₆ alkenylene, or optionally substituted C₂-C₆ alkynylene group, adivalent optionally substituted C₆-C₁₀ aromatic hydrocarbon group, or adivalent optionally substituted saturated or unsaturated C₂-C₁₀heterocyclic or optionally substituted C₁-C₁₀heteroaryl group; R¹⁹ ishydrogen, optionally substituted C₁-C₆ alkyl or optionally substitutedC₃-C₅ cycloalkyl; Y is a bond or a linear or branched optionallysubstituted C₁-C₁₀ alkylene which further optionally has acycloalkylidene structure on one carbon atom, or is optionallysubstituted C₂-C₆ alkenylene, or optionally substituted C₂-C₆ alkynylenegroup; Z is —PO₂—NR³¹R³², —SO₂NR³¹R³², —NR³PO₂—R⁴, or —NR³SO₂—R⁴,wherein R³¹ and R³² are the same or different and each represents ahydrogen atom, optionally substituted C₁-C₆ alkyl group optionallysubstituted with an aryl group, wherein the aryl group, together withthe R³¹ or R³², may form a condensed bicyclic hydrocarbon, or R¹ and R²are taken together with the adjacent nitrogen atom form an optionallysubstituted C₂-C₁₀ heterocyclic group; Z¹ is —PO₂—NR³¹R³² or R³PO₂—R⁴wherein R³¹ and R³² are independently a hydrogen atom, optionallysubstituted C₁-C₆ alkyl group optionally substituted with an aryl group,wherein the aryl group, together with the R³¹ or R³², may form acondensed bicyclic hydrocarbon, or R³¹ and R³² are taken together withthe adjacent nitrogen atom form an optionally substituted C₂-C₁₀heterocyclic group; R³ is hydrogen or optionally substituted C₁-C₆alkyl; and R⁴ is optionally substituted C₆-C₁₀ aryl or an optionallysubstituted C₂-C₁₀ heterocyclic group.
 2. The compound of claim 1,wherein the uracil isostere is an optionally substituted cycloalkyl oroptionally substituted heterocyclyl ring which is monocyclic, bicyclic,tricyclic, or tetracyclic, wherein the ring comprises a moiety selectedfrom —C(═V)—NH—C(═V)—, —C(═V)—CH₂—C(═V)—, optionally substitutedmeta-dihaho phenyl, such as meta-difluoro phenyl,

wherein each V independently is O or S, each R¹ independently ishydrogen, C₁-C₆ alkyl optionally substituted with C₃-C₅ cycloalkyl, orC₃-C₅ cycloalkyl, each R² is independently —OH, —SH, —OR¹, —SR¹, or halowherein R¹ is defined as above, R¹⁰ is hydrogen, R¹², or —O—R¹², whereinR¹² is C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionallysubstituted with 1-3 hydroxy, fluoro, chloro, and amino substituent, R¹¹is hydrogen, halo, R¹² or —O—R¹², wherein R¹² is defined as above, r is1, 2, or 3, each Q¹ and Q² independently are —CH₂—, O, S or an oxidizedform thereof, NH or an oxidized form thereof, or Q¹ and Q² together forma —CH═CH— moiety; provided that Q¹ and Q² are both not O, S or anoxidized form thereof, NH or an oxidized form thereof or a combinationof each thereof; wherein each —CH═, —CH₂—, and —NH— is optionallysubstituted. 3-15. (canceled)
 16. The compound of claim 1 wherein theuracil isostere is:


17. The compound of claim 1, wherein the uracil isostere is:


18. The compound of claim 1 wherein the uracil isostere is:


19. The compound of claim 1 wherein the uracil isostere is:


20. The compound of claim 1 wherein the uracil isostere is:


21. The compound of claim 1, wherein —W—X—Y— is—CH₂—X—SO₂—NH—CH(R^(Y))—, —CH₂—X—SO₂—NH—C(R^(Y))₂—, or—CH₂—X—B—CH₂CR^(Z)R^(W)—X is optionally substituted C₁-C₆ alkylenewherein one of the methylene groups within the alkylene chain isoptionally replaced with an O or S atom, such that X is optionallysubstituted alkylene or a heteroalkylene; B is a optionally substitutedC₃-C₁₀ heteroaryl; R^(Y) an R^(w) are independently hydrogen oroptionally substituted C₁-C₆ alkyl; and R^(z) is hydrogen or hydroxy.22. The compound of claim 1, wherein —W—X—Y— is

wherein Y¹ is CH₂, O or S, X¹⁰ is NH, NCO₂R²⁰, O, or CH₂, R²⁰ is C₁-C₆alkyl optionally substituted with 1-3 C₆-C₁₀ aryl groups, u is 0, 1, 2,3, or 4, and R^(z) is hydroxy or hydrogen, and R^(w) is C₁-C₆ alkyl orhydrogen, and the phenylene and the heteroarylene rings are optionallysubstituted.
 23. The compound of claim 1, wherein Z is:

R⁶ is hydrogen or halo, and R⁷ is optionally substituted C₁-C₁₀ alkyl,optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆alkynyl, optionally substituted C₃-C₅ cycloalkyl, optionally substitutedC₃-C₁₀ heteroaryl, optionally substituted C₃-C₁₀ heterocyclyl, oroptionally substituted phenyl.
 24. A compound, or a tautomer thereof,including any stereoisomer, enantiomer or diastereoisomer, andpharmaceutically acceptable salt of each thereof, wherein the compoundselected from:


25. A compound, or a tautomer thereof, including any stereoisomer,enantiomer or diastereoisomer, or a pharmaceutically acceptable salt ofeach thereof of formula (III):

wherein A is

R¹⁰ is hydrogen, R¹², or —O—R¹², R¹² is C₁-C₆ alkyl, C₂-C₆ alkenyl, orC₂-C₆ alkynyl optionally substituted with 1-3 hydroxy, fluoro, chloro,and amino substituent, R¹¹ is hydrogen, halo, R¹² or —O—R¹², wherein R¹²is defined as above, r is 1, 2, or 3, L¹- is

wherein Y¹ is CH₂, O, S, X¹⁰ is NH, NCO₂R²⁰, O, or CH₂, R²⁰ is C₁-C₆alkyl optionally substituted with 1-3 C₆-C₁₀aryl groups, u is 0, 1, 2,3, or 4, R^(z) is hydroxy or hydrogen, R^(w) is C₁-C₆ alkyl or hydrogen,and the phenylene and the heteroarylene rings are optionallysubstituted, Z is phenyl or a 5 or 6 member heteroaryl substituted withan R⁶ and an R⁶⁰ groups, wherein the R⁶ and the R⁶ are positioned 1,2with respect to each other, R⁶ is hydrogen, optionally substituted C₁-C₆alkoxy, or halo, and R⁶⁰ is —OR⁷ or —NHR⁷R⁷⁰, R⁷ is optionallysubstituted C₁-C₁₀ alkyl, optionally substituted C₂-C₆ alkenyl,optionally substituted C₂-C₆ alkynyl, optionally substituted C₃-C₅cycloalkyl, optionally substituted C₃-C₁₀ heteroaryl, optionallysubstituted C₃-C₁₀ heterocyclyl, or optionally substituted phenyl, andR⁷⁰ is hydrogen or R⁷.
 26. The compound of claim 25, wherein L¹ is


27. The compound of claim 25, wherein L¹ is


28. The compound of claim 25, wherein the uracil isostere is:


29. The compound of claim 25, wherein the uracil isostere is:


30. The compound of claim 25, wherein the uracil isostere is:


31. The compound of claim 25, wherein the uracil isostere is:


32. The compound of claim 25, wherein Z is selected from:

wherein each R⁶ and R⁷ independently are defined as in claim 25 above,each R⁶¹ and R⁶² independently is N or CH, provided that at least one ofR⁶¹ and R⁶² is N, each R⁶³ independently is NR⁷⁰, S, O, and each R⁶⁴independently is N or CH.
 33. The compound of claim 25 of formula:


34. The compound of claim 25, of wherein L¹ is defined as in claim 24above, R⁶ is hydrogen, F, Cl, OMe, or OCF₃, and R⁷ is

wherein t is 1, 2, or
 3. 35. A stereochemically pure enantiomer of acompound of claim 1, or its pharmaceutically acceptable salt. 36.Compound PCI 10586 or pharmaceutically acceptable salt thereof.
 37. Acomposition comprising the compound of claim 1 and a carrier or anexcipient.
 38. The composition of claim 37, wherein the carrier or theexcipient is a pharmaceutically acceptable carrier or excipient.
 39. Amethod of one or more of inhibiting dUTPase or enhancing the efficacy ofa dUTPase directed therapy comprising contacting the dUTPase with thecompound of claim
 1. 40-52. (canceled)
 53. A method of treating adisease whose treatment is impeded by the expression or over expressionof dUTPase, comprising administering to a patient in need of suchtreatment an effective amount of the compound of claim
 1. 54-61.(canceled)
 62. A method of inhibiting the growth of a cancer cellcomprising contacting the cell with an effective amount of the compoundof claim 1 and an effective amount of a dUTPase-directed therapeutic,thereby inhibiting the growth of the cancer cell. 63-75. (canceled) 76.A method of treating a disease in a patient whose treatment is impededby the expression or over expression of dUTPase, comprising: a.screening a cell or tissue sample isolated from the patient; b.determining the expression level of dUTPase in the sample, c.administering to a patient whose sample shows over expression of dUTPasean effective amount of the compound of claim
 1. 77-78. (canceled) 79.The compound of claim 1 of formula:

wherein A is selected from: X¹⁰ is NH, NCO₂R²⁰, O, or CH₂; R²⁰ is C₁-C₆alkyl optionally substituted with 1-3 C₆-C₁₀ aryl groups; u is 0, 1, 2,3, or 4; R¹¹ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynylwherein each alkyl, alkenyl, and alkynyl is optionally substituted with1-3 hydroxy, fluoro, chloro, and amino substituent; R₆₀ is C₁-C₆ alkyl,and r is 1, 2, or
 3. 80. The compound of claim 79, wherein A is:


81. The compound of claim 80, wherein A is selected from:

82-85. (canceled)
 86. The compound of claim 79, selected from:

and a diastereomer or an enantiomer thereof, or a pharmaceuticallyacceptable salt thereof.
 87. A composition comprising the compound ofclaim
 79. 88. The composition of claim 87, wherein the carrier or theexcipient is a pharmaceutically acceptable carrier or excipient.
 89. Amethod of one or more of inhibiting dUTPase or enhancing the efficacy ofa dUTPase directed therapy comprising contacting the dUTPase with thecompound of claim
 79. 90-128. (canceled)